Not all hematopoietic stem cells (HSCs) are alike. They differ in their physical characteristics such as cell cycle status and cell surface marker phenotype, they respond to different extrinsic signals, and they have different lineage outputs following transplantation. The growing body of evidence that supports heterogeneity within HSCs, which constitute the most robust cell fraction at the foundation of the adult hematopoietic system, is currently of great interest and raises questions as to why HSC subtypes exist, how they are generated and whether HSC heterogeneity affects leukemogenesis or treatment options. This Review provides a developmental overview of HSC subtypes during embryonic, fetal and adult stages of hematopoiesis and discusses the possible origins and consequences of HSC heterogeneity. Summary: This Review takes a close look at hematopoietic stem cell heterogeneity during development and in the adult, and discusses several different ways in which this heterogeneity may arise. Hematopoietic stem cells (HSCs) give rise to all cells in the blood lineage through the process of hematopoiesis. In order to do this, HSCs undergo self-renewal, retaining their multipotentiality and thus their function throughout life. While it is convenient to think of HSCs as a single, homogenous population, evidence from recent years does not support this view. The concept that HSCs can be sorted to homogeneity and that each HSC behaves identically in reconstitution assays is under challenge. Clonal HSC transplantations and refined cell sorting have identified HSC subtypes with different functional properties, including differences in repopulation kinetics , cell cyW/W) recipient mice  HSCs showed selective enrichment of only some kinetic subsets of all of the HSC types found in bone marrow. Moreover, daughter HSCs from primary hosts, when transplanted into secondary hosts, followed the same repopulation kinetics as the original transplanted HSC (The Muller-Sieberg group was among the first to define HSC heterogeneity by observing the repopulation kinetics of single HSCs following transplantation into irradiated or cKit receptor tyrosine mutant (ent mice . Statistnted HSC . This re− Lin− cKit+ Sca1+) HSCs by competitive repopulation assays in both primary and secondary recipients, and the results showed that great diversity exists in the repopulating activity between HSC clones , erythroid, platelet/megakaryocyte and lymphoid (B and T cell) lineages. Measurement of mature hematopoietic cell lineage outputs in the peripheral blood at 4-6 months following clonal stem cell transplantation revealed that HSCs fall into several subtypes. These could be retrospectively classified as being ʻmyeloid-biased' , ʻlymphoid-biased'  and ʻmyeloid-lymphoid balanced', based on the measurement of the predominant lineages within the total output of donor-derived HSCs  or based+ subset, and vWF+ HSCs showed higher myeloid and lower lymphoid contributions compared with vWF− HSCs  . PlateleWF− HSCs .Others have classified HSC subtypes into long-, intermediate- and short-term  repopulating HSCs based on the reconstitution kinetics over a period of 24 weeks following clonal HSC transplantation into primary recipients and 20 weeks in secondary recipients . AlthougThe relationship between the LT/IT/ST classification system and the other HSC subtype classifications  was asse− Sca1+ cKit+ CD150+ CD48− , which are normally quiescent in situ in the steady-state bone marrow, are highly proliferative after transplantation and can efficiently repopulate secondary and tertiary recipients. By contrast, cKithigh HSCs have low expansion capacity and reduced repopulating activity in primary recipients and after serial transplantations  . Differesed HSCs . In addised HSCs . Hence, Pax5, Il7r, E2a (Tcf3) and Ikaros (Ikzf1), than myeloid-biased HSCs  may be due to the downregulation of IL7 receptor (IL7R) . Lymphocsed HSCs .in vitro or injection of TGFβ1 into mice in vivo in vitro sed HSCs , were sised HSCs .Altogether, these studies have yielded insight into the some of the intrinsic molecular differences between HSC subtypes.Given the relationship between HSC lineage output, molecular phenotype and responsiveness to signalling pathways, it is of great interest to understand the origins of HSC heterogeneity. There are several different scenarios that might explain how HSC heterogeneity arises, including the influence of distinct HSC niches in the adult bone marrow, a direct lineage progression from one HSC type to another, distinct embryonic origins, and/or different developmental microenvironments . For theAdult HSC function may be related to HSC localisation within the bone marrow niches. Recent studies focusing on the mouse bone marrow niche have demonstrated that the HSC-supportive niches are composed of combinations of highly diverse ‘stromal’ cell types. Cells that comprise these niches include osteoblasts, macrophages, megakaryocytes, sympathetic nervous system cells, endothelial cells, and perivascular and perisinusoidal mesenchymal stromal/stem cells . This imBmpr1a  perivascular cells were depleted, HSCs were also reduced in number and were more quiescent, suggesting a key role for these cells and the chemokine that they secrete in controlling HSC proliferation  and CXCL12 . Conditi numbers . Deletioferation . Whetherferation . Howeverferation . HSC celferation .Finally, HSC localisation in the bone marrow niche can be modulated intrinsically by nuclear factor-like 2 , a master regulator of the oxidative stress response. This factor, expressed by HSCs themselves, acts as a negative regulator of cell cycle progression by partially regulating CXCR4 expression .On their own, local blood perfusion and hypoxia can functionally separate HSC populations in the bone marrow niche .Altogether, these observations suggest that HSC heterogeneity is supported by the high diversity of cell types found in the supportive niches of the bone marrow; however, it remains unknown whether HSC heterogeneity is unique to the bone marrow or whether some HSC subtypes are developmentally determined.During development of the adult hematopoietic system, HSCs are localised in several different microenvironments that are not only supportive but also elicit unique inductive and expansion properties  Fig. 3)Fig. 3). HSCs of the embryo, fetal liver, neonatal bone marrow and adult bone marrow have been studied and compared for properties such cell cycle, self-renewal and lineage output. Almost 100% of HSCs in the E14.5 fetal liver were found to be actively cycling, compared with only ∼10% of adult bone marrow HSCs – indeed, most adult bone marrow HSCs are quiescent . Little As early as the fetal liver stage, HSC subtypes can be found that show heterogeneity in their lineage output. Clonal transplantations of mouse fetal liver HSCs show HSC subtypes similar to those found in the adult bone marrow: lymphoid-deficient balanced and myeloid-deficient were found at E14.5 . InteresSox17 causes severe fetal hematopoietic defects and an absence of definitive HSCs. Conditional deletion of Sox17 during mid-gestation using endothelial-directed Cre [driven by Tie2 (Tek) or VE-cadherin] resulted in lethality at E13.5. When Sox17 was deleted in hematopoietic cells in 2- to 6-day-old neonates using Mx1-Cre, all mice died by 14 days after birth. However, conditional deletion of Sox17 in hematopoietic cells of 6-week-old mice using Mx1-Cre did not affect hematopoiesis. These data suggest that Sox17 is required for the maintenance of neonatal but not adult HSCs, and that Sox17 is a key determinant of the earliest stages of HSC identity. Additional members of the SOX family, Sox7 and Sox18, are also expressed in the dorsal aorta of the mouse embryo and show similar effects in the formation of cell clusters with hematopoietic activity  HSCs, but not in adult HSCs . Sox17 iactivity . Both Soansition . Sox7 anansition . In thatCebpa in Mx1-Cre adult transgenic mice confers a gain of fetal HSC-type characteristics on the bone marrow HSCs, including proliferation ratio and number of repopulating HSCs  hematopoietic gene expression , are expressed throughout HSC ontogeny. Fgd5 is predominantly expressed in HSCs in both adult bone marrow and embryonic hematopoietic sites   , there ion (EHT) . Thus, don (EHT) . Since, Runx1 silencing   . The rap (Cd168) .in vivo evidence to demonstrate that distinct pre-HSCs or imHSCs contribute to HSC types found at later stages. Nonetheless, the asynchronous and graded cellular maturation could explain the heterogeneity of HSCs in the fetal liver. Individual cells might require different microenvironments to mature or may mature at different rates (Despite these interesting findings, there is still no direct nt rates . Based oin vivo (HSC generation in the AGM region occurs in a polarised manner, with HSCs detected only on the ventral side of the aorta . The iniin vivo , initiatin vivo . Notch 1in vivo . A signain vivo .in vivo and in vitro. Based on a BMP activation marker, HSCs generated in the E11 AGM in vivo are of one type: BMP-activated (Two HSC subtypes that differ in activation by the BMP signalling pathway were identified in studies that examined HSC development in the AGM ctivated . Howeverctivated . Furtherin vivo in the E12-E14 fetal liver. Limiting dilution transplantations of E14 fetal liver show that 80-90% of fetal liver HSCs are of the BMP-activated subtype (Smad1/5 knockout experiments to be dispensable for fetal liver HSC activity (The two HSC types that differ in their responsiveness to BMP were also found  subtype . Interesactivity , suggestactivity . In contactivity . Clonal activity .Adult bone marrow HSCs are also controlled by TGFβ signalling . MyeloidThese results highlight the developmental changes that occur in HSCs and suggest that the bone marrow niche might influence HSC subtypes via the specific developmental growth factors that they secrete.in vivo from early developmental stages, and different HSC subtypes can be detected as early as the fetal liver stage. HSC subtypes exhibit measurable functional properties, such as lineage output or self-renewal ability, and can be prospectively enriched based on surface expression levels of CD150 and cKit, or their responsiveness to the BMP or TGFβ developmental signalling pathways. Although as yet unknown, it is likely that gradients of these morphogens drive the emergence of different HSC types. The responsiveness to these signalling pathways is in association with, but not in complete correlation to, lineage output. Intrinsic molecular differences in HSCs from the fetal liver and adult stages have been found, and genes have been associated with specific HSC lineage outputs. However, the molecular networks that regulate the generation of specific HSC subtypes remain unknown, as do the developmental cellular precursors of HSC types. The changing balance and frequencies of HSC subtypes in the embryonic, fetal and adult microenvironments support the notion that distinct developmental niches are present and differentially affect the persistence and representation of specific HSC types and behaviours. Some studies are beginning to show that the maintenance of self-renewal of the HSCs that colonise the fetal liver depends on the interaction with the endothelial and perivascular cells (HSC heterogeneity exists ar cells . Future The physiological relevance of having different HSC types and subtypes is as yet uncertain. It is possible that lineage-biased HSCs in the bone marrow of the adult could confer an advantage during stress conditions or trauma in which the rapid replacement of a specific lineage of hematopoietic cells is required. In blood-associated cancers, some leukemias are of the myeloid type, whereas others are of the lymphoid type. Specific HSC subtypes in the adult or in the fetal stages might therefore serve as the targets of pre-leukemic events, and thus restrict the malignancy to the myeloid or lymphoid lineage. Additionally, the balance between the types of HSCs might change during hematological disease. Whether only HSCs are being mobilised under these conditions is unclear. A recent study proposed that steady-state hematopoiesis is derived from long-lived progenitors, both restricted and multipotent, rather than LT-HSCs . However


Caulobacter crescentus divides asymmetrically to yield two functionally distinct cells: a motile, chemotactic swarmer cell, and a sessile stalked cell with replication and division capabilities. Work from several Caulobacter labs has revealed that differentiation requires concerted regulation by several two-component system (TCS) signaling pathways that are differentially positioned at the poles of the predivisional cell . The strict unidirectional flow from histidine kinase (HK) to the response regulator (RR), observed in most studied TCS, is difficult to reconcile with the notion that information can be transmitted between two or more TCS signaling pathways. In this study, we uncovered a mechanism by which daughter cell fate, which is specified by the DivJ-DivK-PleC system and effectively encoded in the phosphorylation state of the single-domain RR DivK, is communicated to the CckA-ChpT-CtrA signaling pathway that regulates more than 100 genes for polar differentiation, replication initiation and cell division. Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction.Bacteria face complex decisions when initiating developmental events such as sporulation, nodulation, virulence, and asymmetric cell division. These developmental decisions require global changes in genomic readout, and bacteria typically employ intricate (yet poorly understood) signaling networks that enable changes in cell function. The bacterium  Caulobacter's asymmetric cell-division. Among them are the DivJ-DivK-PleC pathway, which establishes cell identity through the unequal partitioning of a kinase (DivJ) at one cell pole and a phosphatase (PleC) at the other cell pole . Differential positioning of these signal transduction proteins upon compartmentalization prior to the completion of cell division enables cell-type specific phosphorylation of the DivK response regulator (RR) . A second critical TCS system, CckA-ChpT-CtrA pathway, is a phosphorelay system that controls activation of the CtrA master transcription regulator CtrA that regulates polar differentiation and cell-cycle progression. An unusual histidine kinase, DivL, was shown by the Laub and Ryan labs to bind DivK~P specifically and inhibit the CckA pathway in vivo by a poorly defined mechanism .More than two decades of research from the labs of Christine Jacobs-Wagner, Michael Laub, Kathleen Ryan, Yves Brun, Patrick Viollier, Sean Crosson, Urs Jenal, Emanuele Biondi, Austin Newton, and our own have revealed critical roles for TCSs in the regulation of Structural analysis in collaboration with Ashley Deacon and Qingping Xu revealed that DivL adopts a HK structural fold, which normally functions by regulating the phosphorylation state of a response regulator. In contrast, we found that DivL does not covalently modify the phosphorylation state of DivK. Instead, we observed that DivL preferentially binds phosphorylated DivK (DivK~P) over the unphosphorylated form. Additionally, the phosphorylation state of DivK~P is significantly stabilized in the presence of DivL. These observations suggest that the non-catalytic kinase output domain of DivL has been uniquely converted into aninput sensory domain that specifically detects the phosphorylated form of the RR DivK.only the kinase region bound similarly to both the unphosphorylated and phosphorylated forms of DivK. Only when the sensory PAS domain was added back to this "kinase-only" DivL mutant did we observe highly specific recognition of DivK~P over unphosphorylated DivK . Taken together, our findings suggest that the RR docking region and the attached sensory domain cooperate to not only specify the RR binding partner but also to selectively detect covalent modifications made to the cognate RR . We propose that these observations could have broad implications for how TCS kinases can influence whether a protein containing a HK-fold functions as a kinase by promoting unphosphorylated RR binding or as a phosphatase by promoting RR~P binding. Future structural characterization will dissect whether the PAS sensory domain influence on the HK-RR interaction occurs by reconfiguring RR docking region or by directly making contacts with the RR.A key question we addressed in this study was how DivL specifically binds DivK~P over DivK. Many laboratories have convincingly demonstrated that surface-exposed residues within the “RR docking module” of a TCS HK specify the binding between a given HK and its cognate RR , and we indeed found that a set of RR docking module point mutations in DivL disrupt DivK binding and, consequently, result in a loss of cell viability. However, the contribution of PAS family sensor domains to the HK-RR interaction is less well understood. We critically observed that DivL variants containing in vivo DivL functions as a cell-fate switch by inhibiting CckA signaling activity when bound to DivK~P. To understand how DivK~P binding imparts a switch-like function to DivL  we examined a DivL structural region that is broadly important for allosteric kinase activation. Despite its physical distance from the RR docking module, we found that a point mutation within this DivL allosteric regulatory region eliminated binding of DivK~P to DivL in a PAS domain dependent manner. From these experiments, we propose that DivK~P binding to DivL drives a conformational change within the DivL PAS sensory domain to allow a DivL-CckA interaction that inhibits the CckA signaling pathway . Therefore, unlike conventional HK-RR signaling pathways, the signaling information in this case flows in reverse direction - from the RR DivK to the pseudokinase DivL . Critically, this signal flow reversal connects two TCS pathways that culminate in the regulation of differential gene expression in daughter cells that result from asymmetric cell division. This finding raises another important question: do other bacterial TCS signaling systems take advantage of reversals in signal flow?Work from the Laub and Ryan labs revealed that While eukaryotic pseudokinases have been demonstrated to play crucial roles in signaling, few studies of bacterial TCS pseudokinases have been reported. Loss of DivL kinase catalytic functions allows other functional capabilities within the HK-fold to emerge. We have shown that the pseudokinase DivL, with a tyrosine replacing the histidine, retains one HK-fold function in that it binds a cognate RR; our results also identified a second conserved HK function, namely the ability to undergo switch-like conformational change upon binding to a phosphorylated RR. Conservation of these two functions uniquely re-wires DivL to connect two TCS signaling pathways. More generally, the pseudokinase DivL shares broad characteristics with eukaryotic pseudokinases: (1) DivL modulates the signaling activity of another functional kinase (CckA), and (2) other work led by Antonio Iniesta has shown that DivL is necessary to recruit CckA to the signaling hub at the new cell pole. Approximately 1% of sequenced TCS HKs contain an amino acid substitution at the phosphorylatable histidine site , raising the possibility that bacteria utilize pseudokinases where simple TCS pathways offer inadequate processing capabilities.Caulobacter genome alone encodes more than a dozen multi-PAS-domain sensor kinases. Multiple PAS sensory domains offer the potential to integrate multiple signals, although very little is known about how multi-domain sensors integrate information. Are these sensory domains independent of one another, functioning as OR logic gates, or do they work cooperatively as AND gates? Given that PAS sensory domains are commonly involved in protein-protein interactions, it will be interesting to determine whether signaling cross-talk occurs between other HK sensory domains. Future discovery of bacterial pseudokinases and dissections of HK sensory domain mechanisms will help illuminate how bacteria have modified and adapted the canonical HK-fold to generate complex signaling circuits that underlie critical developmental decisions.A second interesting trend in bacterial developmental TCS signaling pathways is the prevalence of histidine or pseudohistidine kinases with multi-PAS domain sensors. For example, the


Late seroma formation is a rare complication after implant-based breast enlargement surgery and even less frequent after implant removal. This case report presents a case of painful recurrent seroma formation after the removal of a ruptured Poly Implants Prothèse implant.A 52-year-old patient presented herself in our clinic with a clinical history of recurrent unilateral seroma of the right breast over a period of 8 years after the initial unilateral implant removal. Removal of the remaining implant and complete bilateral capsulectomy was performed. Intraoperative findings revealed a macroscopically thickened capsule with signs of chronic inflammation on the affected side. The clinical history and the macroscopic appearance of the capsule demanded histopathological exclusion of a possible anaplastic large cell lymphoma.Histopathological and microbiological analysis of the capsule and encapsulated material revealed no signs of malignancy or infection. Immediate soft tissue reconstruction of the breast was successfully performed using autologous fat transfer. An aesthetically satisfying result regarding symmetry and volume was achieved, and no further seroma formation was observed within a 6-month follow-up period.Level of evidence: V, Case Report. The exact rate of implant-related complications is unknown.Although seroma formation is clinically perceived as a well-known complication after implant removal, especially if the capsule is left unmodified in situ, peer-reviewed scientific literature on this subject is rare.Various publications have defined late seroma as a predominant serous accumulation of periprosthetic liquid (exudate or effusion) within the implant capsule developing at least 12 months after the implantation.Vascular and/or lymphatic leakage occurring in comorbid conditions such as chronic inflammation due to subclinical bacterial infection or a local inflammatory response, leading to the release of mediators increasing, eventually, the interstitial fluid drainage.Recurrent trauma with synovial metaplasia due to shearing forces and micromotions between the implant and surrounding tissues.Idiopathic reasons related to reconstructive surgery after malignant diseases.+, with anaplastic lymphoma kinase 1 (ALK-1) negative T cell neoplasms accounting for 0.5% of all breast cancers.Furthermore, late seroma seems to be associated with specific entities of anaplastic large cell lymphoma (ALCL), a rare type of non-Hodgkin lymphoma. These types of ALCL are CD30In recent times, two distinct clinical pathological entities with different prognostic outcomes have been described:In-situ implant-associated ALCLs have an indolent clinical course and tend to show complete remission after complete removal of the capsule.Infiltrative ALCLs seem to have a more aggressive clinical course with a less favorable outcome even after additional therapy.It is important to mention that the clinical symptoms of in situ ALCLs typically do not differ from those of a late seroma.A 52-year-old patient presented herself at our clinic with a history of recurring seroma of the right breast over a period of 8 years. Her surgical records showed bilateral epipectoral breast augmentation in 2002 with cohesive Silicone Gel implants manufactured by PIP . Clinical history revealed a unilateral implant rupture confirmed by mammography and recurrent episodes of local pain and swelling of the corresponding lymph nodes, leading to unilateral implant removal without capsulectomy in 2008.Painful seroma formation recurred in the right breast after several years and was treated repeatedly by transcutaneous needle aspiration. Clinical records mention a fluid of a pale yellowish color without cell debris. Clinical records or anamnestic history could not confirm any cytological or bacteriological tests.A primary physical examination at our clinic showed the right breast had hardened and was painful at palpation. An ultrasound examination revealed a large intracapsular mass with liquid and solid portions. No clinical signs of infection were noticed; the axillary, subclavian, and parasternal lymph nodes were unsuspicious. A considerable asymmetry of the breasts, with the right breast being significantly bigger than the left, was noted andB.Suggested preoperative needle aspiration with cytological and microbiological testing was declined by the patient with reference to her year-long, painful history of symptomatic treatment by needle aspiration; the patient wished for a one-stage solution and was, therefore, scheduled for complete bilateral capsulectomy, removal of the remaining implant on the left side, and reconstruction with autologous fat transfer.The capsule on the left side was removed en bloc with the remaining implant and showed no signs of capsular contracture, double capsule, or implant rupture. After the removal of the capsule, the implant could be identified as a textured 380 cc round and high profile device manufactured by PIP. Explanation data was entered into the Spanish Registry.An intact capsule with macroscopic signs of chronic inflammation, hypervascularity of the outer surface, and stiff, thickened walls was found on the right side. No pericapsular liquid was present. To facilitate en bloc capsulectomy on the right side, transcapsular needle aspiration was performed under direct vision removing 600cc of dark, serous liquid. After en bloc capsulectomy through a periareolar access, the capsule was opened and showed a large amount of a brownish, hematofibrous content without macroscopic evidence of intact, tissue-like structures andB.Immediate volume reconstruction was performed using WAL-assisted  autologous fat transfer from the abdomen. Using a multichannel, multilayer technique , 880 cc of fat was injected into the right breast and 720 cc into the left breast. To reduce the large skin excess resulting from the seroma-induced soft tissue expansion andD wPostoperative recovery was complication-free. No signs of infection, fat necrosis, or recurrent seroma were observed during the 6-month follow-up period.An acceptable aesthetic result was achieved with a single-session autologous fat transfer. The high-volume transfer was feasible due to the largely extended tissue matrix resulting from the tissue expansion effect of the recurrent seroma on the right side and the implant on the left side. Three-month and 6-month postoperative follow-ups –F shoHistological examination of the capsule after embedding and hematoxylin and eosin (HE) and periodic acid–Schiff staining showed tissue changes compatible with a chronic inflammatory reaction characterized by large quantities of inflammatory cells and an abundant presence of foreign body granuloma .Staphylococcus aureus.The presence of malignant cells or especially cells compatible with an entity of ALCL could not be confirmed histologically in the capsule tissue or its content. Neither cytological analysis of the aspirate in HE nor Papanicolaou and Giemsa staining detected atypical cells. Microbiological examination of the needle aspirate showed the presence of a multisensibleKi-1/Ber-H2 (CD 30) and ALK. They were positive for CD 5, CD20/L26/PAN-B, CD 79a, and CD3/UCHL-1/PAN-T, indicating a lymphoid inflammatory process without atypical cell proliferation.Aspirate and capsule were sent for immunohistochemical testing regarding hematopathological markers correlating with ALCL. Results were negative forThe authors conducted a PubMed database query to find similar cases and possible causes described in literature for the recurrence of late seroma after implant removal. The terms used for the query were  AND seroma; (PIP implant) AND seroma;  AND seroma;  AND (periprosthetic fluid), (implant rupture) AND seroma.Apart from a study published in Radiology,An increasing number of meta-analyses shows the possibility that ALCL, in cases with persistent, implant-related late seroma, may not be as rare as previously stated in literature, although the direct causality between textured breast implants and ALCL is not yet proven.One of the four case reports similar to our case describes an ALCL-related malignant effusion. In this case, the patient suffered from recurrent fluid formation within the capsule after implant removal. Repeated biopsies and needle aspirations of the fluid failed to detect ALCL. Only after capsulectomy and complete histological examination, could ALCL be histologically confirmed.These recommendations—the intraoperative findings described above and the long clinical history of recurrent seroma—demanded histopathological and immunohistochemical exclusion of ALCL in our case.S. aureusin our case is interesting within the context of the current discussion literature of possible causality between textured implants, biofilm formation, and ALCL. In a study by Hu et al, the numbers of B and T cells showed a linear correlation with the number of detected bacteria in implant capsules in humans.The microbiological finding of capsule contamination withIn our opinion, additional microbiological testing should be performed in all cases of late seroma. In clinically highly suspicious cases for ALCL, immunohistochemical testing of the capsule and its content should be performed even if the histopathological tests do not detect any signs for ALCL.Immediate volume reconstruction through autologous fat transfer was performed. The authors discussed this procedure preoperatively and decided in favor of immediate reconstruction, despite the intraoperative findings. Clinically, the ongoing process was limited to the capsule and its content. In case of an intracapsular or strictly capsular ALCL, complete capsulectomy can be a definite treatment. Further chemotherapy can be spared.Other treatment options in our case with a severe lack of soft tissue density would have been an open wound treatment or the implantation of a placeholder, aiming to fill the immense wound cavity until the completion of the definite histological analysis. Later, subsequent secondary breast reconstruction through fat grafting, with implants or even through autologous breast reconstruction via deep inferior epigastric perforator flap or analogue techniques could have been performed.p < 0.001) in rupture rates between implants inserted prior to 2003 and those inserted from 2003 was demonstrated.The rupture rate of PIP implants was found to be 35.2% per patient and 21.3% per implant over a mean implantation period of 7.8 years by one group. A statistical difference (S. aureusthat could be the cause a chronic low-grade infection, but due to multiple transcutaneous needle aspirations of the seroma over the last year it remains unclear if perioperative implant contamination or secondary contamination is the source of infection.In the described case, the capsule showed macroscopic signs of chronic inflammation, tissue hardening, and capsular contraction. As the patient had complained about discomfort and swollen axillary lymph nodes before implant removal in 2008, chronic low-grade infection could also be a possible pathomechanism for the recurrent seroma. In the case of an existing periprosthetic biofilm in 2008, implant removal alone would probably not have eliminated the bacterial load,A study by Soo et al in 1995 on 84 patients showed intracapsular seroma formation after implant removal in six cases, while no seroma formation could be observed in patients who underwent complete caspulectomy. Five other case reports also mentioned seroma formation within the capsule.The elimination of dead space after the removal of any implant or tissue is generally recommended for the prevention of seroma and is especially important in case of inflamed capsules. The hardened tissue may prevent the fusion of the anterior and posterior layer and, thereby, leave space for the formation of a potential seroma.The exact cause of recurrent seroma formation in the described case remains unclear. Further investigation is needed to identify the risk factors behind recurrent seroma after implant removal without complete capsulectomy. Complete capsulectomy should be performed in cases with confirmed implant rupture, excessive silicone bleeding, recurrent seroma formation, histopathological changes of the capsule, or proven bacterial load.Since 1997, only 173 cases have been identified so far in the world literature review.As implant removal and complete capsulectomy are considered the only local treatment so far, volume restoration with fat grafting seems to be a valid reconstructive option. Taking advantage of the tissue expansion caused by the implants, one single session of high-volume fat grafting may be sufficient to restore an adequate volume and to achieve an acceptable aesthetic outcome even in breasts with notable skin excess.


Like other organisms, plants have endogenous biological clocks that enable them to organize their metabolic, physiological, and developmental processes. The representative biological clock is the circadian system that regulates daily (24-h) rhythms. Circadian-regulated changes in growth have been observed in numerous plants. Evidence from many recent studies indicates that the circadian clock regulates a multitude of factors that affect plant metabolites, especially emitted volatiles that have important ecological functions. Here, we review recent progress in research on plant volatiles showing rhythmic emission under the regulation of the circadian clock, and on how the circadian clock controls the rhythmic emission of plant volatiles. We also discuss the potential impact of other factors on the circadian rhythmic emission of plant volatiles. Plants synthesize and emit a variety of volatile organic compounds, which have ecological or physiological functions ,3,4,5,6.The circadian clock is a biological oscillator that maintains a 24-h rhythm under normal environmental conditions . CircadiOnce released into the air, volatiles move freely and are difficult to collect. In addition, the emission of many volatiles is variable. Therefore, to precisely analyze emitted volatiles, techniques to collect these compounds should be effective and efficient. Here, we summarize two widely used collection methods A,B. The Studies using these new and sophisticated sampling techniques have markedly increased our understanding of the rhythmic emission of plant volatiles in recent years. Rhythmic emission occurs under direct regulation by light or by the endogenous circadian clock C 9]. Th. Th9]. TTo date, more than 1700 volatile compounds have been isolated from more than 90 plant families, containing almost 1% of all plant specialized metabolites identified so far . During Antirrhinum majus cv. Maryland True Pink), petunia (Petunia hybrida cv. Mitchell), and tobacco (Nicotiana suaveolens) during two normal light/dark cycles, maximum emission was detected in the three different species during a 24-h period, showing a rhythmic fluctuation [Elaeis guineensis) and Grey poplar (Populus × canescens) were shown to be under strong circadian control [E)-β-ocimene) from snapdragon flowers [Volatiles emission is highly regulated, and is restricted to specific times of the day in many plant species. Several recent studies have significantly advanced our knowledge of rhythmic emissions. ctuation ,37. Simi control ,39. Diff control ,40. The  flowers ,41.lipoxygenase (LOX), has been linked to circadian regulation. Compared with volatile fatty acid derivatives, there are more studies investigating the underlying mechanisms of circadian rhythmic emission of volatile terpenes and VPBs  transcript levels continued to fluctuate with a circadian rhythm, resulting in a diurnal pattern of β-pinene content and emission [PhCCD1, which encodes carotenoid cleavage dioxygenase (CCD), the enzyme responsible for β-ionone formation. This result indicated that circadian modulation of PhCCD1 controlled β-ionone emission [Stephanotis floribunda, the expression level of SAMT showed a rhythmic oscillation that coordinated with the emission of MeSA from flowers, while the SAMT protein levels did not show rhythmic changes [isoprene synthase (ISPS), showed circadian variations in its transcript levels [Stephanotis flowers, and peak activity coincided with nocturnal MeSA emission [Several studies have shown that the rhythmic expression of genes encoding enzymes involved in the final steps of volatiles biosynthesis play a role in circadian control. When emission . In petuemission . Salicyl changes . A similt levels . Howevert levels . Enzyme emission . These sd-xylulose-5-phosphate synthase (DXPS), which catalyzes the formation of DXP, showed a diurnal oscillation pattern that strongly correlated with the rate of the MEP pathway  and abiotic factors  affect the emission of plant volatiles. Most studies on plant volatiles emission have focused on one or multiple external stresses. Less attention has been paid to the effects of interactions between external stresses and the endogenous circadian clock on the emission of plant volatiles.Phaseolus lunatus) is an example of the effect of an interaction between biotic stress (insect attack) and the circadian clock on plant volatiles emission. Arimura et al. investigated whether herbivore-induced emission of β-ocimene from P. lunatus leaves was controlled by the circadian clock [PlOS, which encodes ocimene synthase, was shown to be regulated by the endogenous circadian clock, jasmonic acid, and light. In maize, ZmLOX10 was shown to be induced by jasmonate and herbivores for defense against insect attack [ZmLOX10 was regulated by the circadian clock at a transcriptional level [ZmLOX10 was proposed to be a mechanistic link for the circadian emission of GLVs, but there was no direct evidence for the circadian emission of herbivore-induced GLVs. In some cases, therefore, genes involved in the formation and emission of herbivore-induced plant volatiles are regulated transcriptionally by the circadian clock, but this does not necessarily lead to circadian emission of herbivore-induced plant volatiles.The emission of β-ocimene from lima bean  [Van Moerkercke et al. proposed that the circadian clock is responsible for rhythmic volatiles emission in petunia that produce VPBs as the major volatile , and they/night) . Nonethey/night) .1Not all plant volatiles show rhythmic emission regulated by the circadian clock. In addition, not all plants and not all plant tissues show such rhythmic emissions. Why are some plant volatiles rhythmically released and others not? Why is the emission of some plant volatiles controlled by endogenous circadian mechanisms while the emission of others is controlled by external factors such as light or temperature?2Many studies on the regulation of the rhythmic emission of plant volatiles by the circadian clock focus on downstream pathways or the final step in volatile biosynthesis. Is there a unified mechanism of action for the circadian clock to mediate the rhythmic emission of plant volatiles? What are the upstream signaling events that control circadian clock-mediated rhythmic emission?3In most cases, external factors such as insect attack, high temperature, or light readily induce the transient emission  of plant volatiles, some of which can protect plants. Why do plants regulate rhythmic emission of plant volatiles through the endogenous circadian clock? What are the benefits for plant growth and development? Besides ecological functions such as interaction with insects, do volatiles have physiological functions in plants?4Plants are exposed to many environmental factors/stresses. Are there interactions between these environmental factors and regulation of the endogenous circadian clock? If endogenous circadian clock regulation is relatively independent, how do plants maintain the stability of endogenous circadian clock regulation?In this review, we summarize the current knowledge of circadian clock-mediated rhythmic emission of plant volatiles, although few studies have focused directly on this topic. The huge variety of volatiles emitted from plants and their complex and diverse emission patterns suggest that there is still much to learn. Several important questions should be addressed in future studies:


Here, we report our experience with a case of severe biliary bleeding due to a hepatic arterial pseudoaneurysm that had developed 1 year after endoscopic biliary plastic stent insertion. The patient, a 78-year-old woman, presented with hematemesis and obstructive jaundice. Ruptured hepatic arterial pseudoaneurysm was diagnosed, which was suspected to have been caused by long-term placement of an endoscopic retrograde biliary drainage (ERBD) stent. This episode of biliary bleeding was successfully treated by transarterial embolization (TAE). Pseudoaneurysm leading to hemobilia is a rare but potentially fatal complication in patients with long-term placement of ERBD. TAE is a minimally invasive procedure that offers effective treatment for biliary bleeding. Core tip: Biliary bleeding after endoscopic pigtail plastic stent insertion is a rare but potentially fatal complication. Transarterial embolization (TAE) is a minimally invasive procedure that offers effective treatment for pseudoaneurysm. We report here a case of biliary bleeding caused by long-term placement of a pigtail plastic stent, which was inserted without removal of common bile duct stones due to the patient’s age; the TAE treatment was successful. This case report will help similar patients yet to be encountered but likely to increase in number due to ageing of the world’s population.Biliary bleeding is an uncommon cause of upper gastrointestinal bleeding and biliary tract obstruction. Biliary stent-related biliary bleeding that occurs after an endoscopic procedure has been reported less often than biliary bleeding related to the percutaneous transhepatic procedure; however, its incidence has been increasing due to the general increase in endoscopic interventions. Transarterial embolization (TAE) is an effective treatment for traumatic pseudoaneurysm of visceral arteries. We describe, herein, a patient with gastrointestinal bleeding and biliary tract obstruction due to intrabiliary rupture of hepatic arterial pseudoaneurysm, which was successfully treated by TAE and percutaneous transhepatic biliary drainage (PTBD).A 78-year-old woman.Hematemesis.Thirty years before the current admission, the patient had undergone left hepatectomy, cholecystectomy and choledochectomy for intrahepatic and common bile duct (CBD) stones, in conjunction with Roux-en-Y and biliary reconstruction. At 1 year prior to the current admission, the patient had developed acute obstructive cholangitis due to CBD stones and had subsequently undergone single-balloon endoscopy. During that procedure, a 7 Fr pigtail type plastic biliary stent had been inserted into the CBD, which was already dilated due to the previous biliary reconstruction. The proximal edge of the plastic stent had been placed, coiled around, in the CBD improperly  and tachycardia (heart rate of 99 bpm); her blood pressure was 14.4/5.9 kPa and her respiration rate was 20/min. She had no abdominal pain. Laboratory findings were as follows; hemoglobin of 9.6 g/dL ; aspartate aminotransferase/alanine aminotransferase of 155/58 IU/L ; alkaline phosphatase of 1208 IU/L ; total bilirubin of 1.8 mg/dL ; C-reactive protein of 2.6 mg/dL ; white blood cell count of 10180/μL . Non-enhanced computed tomography (CT) revealed dilation of the CBD, as well as high-density masses in the bile duct and in the afferent intestinal loop. Single-balloon endoscopy of the small intestine revealed copious fresh coagulum in the afferent loop, but the site of bleeding was not detected. Contrast-enhanced CT showed 13 mm × 10 mm pseudoaneurysm of the hepatic artery, proximal to the edge of the plastic biliary stent  revealed an aneurysm at A8  with an arterio-biliary fistula  and percutaneous transhepatic biliary drainage.Although biliary hemorrhage is relatively common after surgery, percutaneous transhepatic procedures and placement of self-expanding metallic biliary stents, it is rare after insertion of a plastic stent.The Couinaud classification is used to describe functional liver anatomy. It has emerged as the preferred anatomy classification system since it divides the liver into eight independent functional units.The pigtail plastic stent is often used for temporary biliary drainage. However, it is important to remember that this stent type can cause fatal bleeding. TAE is a more effective and less invasive treatment for biliary bleeding than surgery.Although trauma caused by the proximal tip of the pigtail stent is highly suspicious, the authors explain that there might be other additional factors for the formation of the hepatic pseudoaneurysm.


Stromal-derived intratumoural heterogeneity (ITH) has been shown to undermine molecular stratification of patients into appropriate prognostic/predictive subgroups. Here, using several clinically relevant colorectal cancer (CRC) gene expression signatures, we assessed the susceptibility of these signatures to the confounding effects of ITH using gene expression microarray data obtained from multiple tumour regions of a cohort of 24 patients, including central tumour, the tumour invasive front and lymph node metastasis. Sample clustering alongside correlative assessment revealed variation in the ability of each signature to cluster samples according to patient-of-origin rather than region-of-origin within the multi-region dataset. Signatures focused on cancer-cell intrinsic gene expression were found to produce more clinically useful, patient-centred classifiers, as exemplified by the CRC intrinsic signature (CRIS), which robustly clustered samples by patient-of-origin rather than region-of-origin. These findings highlight the potential of cancer-cell intrinsic signatures to reliably stratify CRC patients by minimising the confounding effects of stromal-derived ITH. Tumour expression profiling is currently used for prognostic and predictive purposes without taking into account the intra patient heterogeneity. Here the authors show that cancer cell specific signatures overcome the tumour heterogeneity effect and result in better classification of colorectal cancer patients. The application of transcriptional gene signatures to stratify tumours into prognostic and predictive subtypes has evolved rapidly since the original landmark studies demonstrated the clinical utility of this approach in breast cancer1Recently, an international colorectal cancer subtyping consortium, tasked with defining the transcriptional landscape of CRC, published their assignment of four consensus molecular subtypes in CRC (CMS1-4)8By evaluating differentially expressed genes between the central tumour (CT) and invasive front (IF) regions of primary colorectal tumours, we previously demonstrated that stromal-derived ITH can be a major confounder of transcriptomics-based CMS patient subtypingWe have previously demonstrated how stromal-derived gene expression could undermine patient classification when using a gene signature associated with the recently published CRC CMSet al.et al.et al.et al.n=24) and invasive front   . To incl4) et al.. Using t4) et al.. In addi4) et al.. In addi4) et al.. The sten=24, total dataset n=72). Utilizing this cohort, we then employed a semi-supervised analysis of each signature, using hierarchical clustering of region-specific samples from both the primary tumour CT and IF, alongside the LN metastasis samples. This analysis confirmed the ability of our previously published stromal-associated 30 gene signature to identify regions-of-origin rather than patient-of-origin, with similar results observed for the stem-like CMS4 classifier . To allow a quantitative comparison of both the intra- and inter-patient similarities for each signature, we implemented an additional normalization step in this analysis (detailed in the Pearson similarity section of the Methods section), by assessing the correlation between samples specifically from the same patient, compared to samples from different patients . Using tBRAF mutational status using gene expression data from the PETACCGiven the results of the clustering and similarity analyses, we hypothesized that the level of performance observed for each signature would be relative to the proportion of stromal and epithelial genes in each classifier. We also proposed that the Popovici signature genes would be predominantly epithelial tumour cell derived, given the superior performance of this signature in our study. The Popovici signature was developed by examining the transcriptional profile associated with epithelial P<0.0001, Tukey's multiple comparison test P<0.05, These results further reinforced the findings of our previous work, in which we reported that cancer-cell extrinsic genes can confound transcriptomics-based patient classification signaturesTo directly compare the patient classification results using the published methodologies for both the CRIS and CMS classifiers, we performed sample classification with the random-forest CMS classifier algorithm, alongside the CRIS classifier, which uses a nearest template prediction (NTP) classifier, on our complete cohort. We observed that while CMS classification results in concordant assignment of 9/24 (38%) of patient-matched CT and IF samples, the CRIS classifier concordantly assigns 17/24 (71%) of patient-matched CT and IF samples . More deFurther comparison of the CRIS signature using the patient similarity normalized index as before , indicatThe potential clinical utility of both the Popovici and CRIS signatures for patient classification is further reinforced by their ability to maintain patient-specific clustering throughout this step-wise analysis, with only a minimal decline in their efficiency as the stringency increases . By plotWe and others have previously demonstrated how transcriptional-based patient classifiers, such as the CMS89Unsupervised clustering of large transcriptomic array data will generally cluster samples according to patient-of-origin. However, classification of patient samples into prognostic/predictive subtypes is generally dependent on small pre-defined gene signatures that can themselves be confounded by region-of-origin of the tissue. When patient stratification is distilled to this level of granularity, subtle changes in the global gene expression profiles at different regions can have profound implication for subtype assignment. Similar to results with our 30 gene signature, the stem-like CMS4 classifier is confounded by region-of-origin when attempting to classify individual patient samples. Signatures such as the Jorissen and Eschrich signatures11In contrast, the Popovici signatureLarge individual and collaborative efforts aimed at defining CRC molecular subtypes have confirmed the important role that the stroma plays in patient stratification and prognosis7192021222425et al.4The platforms used in the generation of the gene signatures in this study include Affymetrix and custom cDNA arrays, alongside next generation sequencing (NGS) technology. Inevitably, when comparing the utility of these signatures, there will be some cases when individual genes/probes are not universally represented across all platforms, resulting in gene dropout. To ensure that this dropout was minimized, we utilized consensus ‘core genes' for the signatures  and principal components analysis (PCA) to assess the quality of the data before analysis. The distribution of sample data  should approximate a normal distribution and should be free of outliers, demonstrated through hierarchical clustering and decomposition techniques (PCA and/or MDS). Hierarchical clustering (condition tree) shows the relationships among the expression levels of samples, allowing identification of any spurious samples. From the initial QC analyses of the microarray data, we identified 2 samples as outliers from the PCA and clustering tree methods  which weet al.https://github.com/Sage-Bionetworks/crcsc) and scripts executed in R studio version 3.3.1. The colorectal intrinsic subtypes (CRIS) were determined using the nearest template prediction (NTP) classifier, available from GenePattern (https://genepattern.broadinstitute.org/gp/pages/login.jsf). The thresholds used for statistical significance were generated using Benjamini-Hochberg–corrected false discovery rate (BH.FDR) <0.2 (ref. The consensus molecular subtype (CMS) for each region profiled was determined using the random forest classifier described by Guinney 0.2 ref. .ftp://ftp.ebi.ac.uk/pub/databases/genenames/new/tsv/hgnc_complete_set.txt)To avoid variations inherent during probeset-to-gene annotation conversion from signatures developed using different platforms, we utilized previously published consensus gene lists for each signature to be evaluated, as implemented by Sanz-Pamplona and colleaguesIn addition, we have used a combination of Entrez ID, Unigene, HUGO and Refseq to comprehensively update the annotation file for the DSA array. Probes without Entrez Gene IDs that had a RefSeq or Unigene ID in the original annotation were assigned an Entrez Gene ID using the NCBI Batch Entrez tool. Probes that hadn't any identifiers apart from a gene symbol were updated by matching that gene symbol to a previous gene symbol or alias found in the hgnc_complete_set file. Despite these efforts, inevitably no match was found for a small number of genes in some signatures and they were lost for subsequent analysis. The overall levels of gene dropout were minimal; the signature with the highest dropout  was due to a loss of only three genes missing from the core gene signature. Given this limitation, the signatures used in our study can only serve as a representation of the original signatures. Importantly however, our study is focussed on dissecting the cellular source of the core genes related to their ability to robustly cluster patient samples to outline parameters that could potentially improve future signature/classifier design. Each individual signature, in its original form, has demonstrated prognostic value and was selected as they represent the most clinically relevant and widely employed signatures to support the aims and findings in this study.The 30 gene signature was previously generatedet al.n=30), poor <36 months survival (n=48) across Dukes stage B, C and D. Three adenoma samples were included within the good prognostic group. Transcription profiles were generated on cDNA arrays. Using a leave-one-out cross validation approach, a 43 gene signature was developed  or no recurrence (n=143). Transcriptional data was generated on the Almac Colorectal Cancer DSA platform. Using cross validation, a 634 probeset signature was developed which identified a prognostic subtype in stage II, which was further validated in an independent cohort of 144 samples. This signature has subsequently been validated in an additional stage II clinical trial cohortThe Kennedy et al.The Popovici The colorectal intrinsic signature (CRIS)For all analyses, we have used the gene sets as detailed in As outlined above, patients were assigned an alphabetical label A–Y; patient sample M was removed as an outlier before analysis. Divisive analysis clustering (DIANA) was performed on the expression values of the patient matched IF and CT samples, ascertaining to each of the gene signatures on default settings. This was completed using the ‘cluster' package in R statistical environment (v3.2.3). The DIANA method is particularly suited to test inter-patient heterogeneity in sample pairs, as it continuously splits samples into two clusters until it reaches single samples, which allows an assessment of both the final patient clustering and the extent to which an individual signature could be undermined by stromal derived transcriptional ITH. Resulting dendrograms were assessed for patient clustering and values were tabulated.Partek Genomics Suite software, version 6.6; 2016 Partek, Inc., St Louis, MO, USA., was used for independent dataset analysis. For the purpose of clustering, data matrices were standardized to the median value of probe sets expression. Standardization of the data allows for comparison of expression levels for different probe sets. Following standardization, two-dimensional hierarchical clustering was performed (samples × probe sets/genes). Euclidean distance was used to calculate the distance matrix, a multidimensional matrix representing the distance from each data point (probe set-sample pair) to all the other data points. Ward's linkage method was subsequently applied to join samples and genes together, with the minimum variance, to find compact clusters based on the calculated distance matrix.The Pearson correlation coefficient was used to define the ratio between the covariance and the standard deviation of multi-region samples for each individual patient. By generating a score for each sample compared to each other sample, this method allowed us to build a matrix based on an enumeration of the similarities of all three samples  for each individual patient. Increased scores indicate that samples display a higher similarity with other matched region-specific samples from the same patient. As the standard Pearson method allows direct correlation of one sample to another, we wished to test if each individual patient score was higher than that observed across all of the samples. To this end, we used a normalized method, which calculates the relative similarity between the three samples from the same patient, compared to their similarity to samples from all other patients within our dataset, from a score of 0 (no increased correlation of patient matched samples compared to samples from different patients) to 1 .We used sequential analyses to give an assessment of the ability of each signature to both cluster patient samples into the same higher-order prognostic/predictive subtype, followed by the ability of the signature to robustly differentiate and cluster primary and metastatic samples according to patient of origin. The various signatures indicated above were used to cluster the data, using hclust, with Ward's linkage and Euclidean distance metric. The resultant dendrogram was then analysed using the cutree function to extract the group membership, as the number of groups is sequentially increased, from 1 to 24 (the number of patients). At each level, the Patient Group Overall Ratio (PGOR) was calculated as: PGOR=Number of Patients Grouped in the Same Cluster/Total Number of Patients,that is, the PGOR=1 if all samples for all patients are found in the same cluster at a particular level, and PGOR=0 if none of the samples group together consistently. The evolution of the PGOR was plotted against the number of clusters, showing the consistency of patient clustering.http://www.ncbi.nlm.nih.gov/geo/) and is available under accession numbers GSE95109 and GPL23083. For testing of the cell lineage-specific source of the transcripts, gene expression profiles from an independent CRC dataset were downloaded from NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE39396. The GSE39396 dataset contains microarray profiles from fresh colorectal specimens where FACS has been used to divide cells into specific endothelial (CD45+EPCAM−CD31−FAP−), epithelial (CD45− EPCAM+CD31−FAP−), leukocyte (CD45−EPCAM−CD31+FAP−) and fibroblast (CD45−EPCAM−CD31−FAP+) populations before microarray profiling. Plots based on transcriptional data were plotted using GraphPad Prism version 5.03 for Windows, GraphPad Software, La Jolla CA, USA, www.graphpad.com. In addition, for the comparison of CRC sample classification by the Sadanandam signatureOur transcriptional data and updated annotation files, alongside patient and region identifiers has been uploaded to the NCBI Gene Expression Omnibus (GEO) repository .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Table30 gene signatureEschrich et al. signatureJorissen et al. signatureSadanandam et al. signatureKennedy et al. signaturePopovici et al. signatureColorectal intrinsic signature (CRIS) signature


Furthermore, noninvasive imaging results were validated with in vivo and ex-vivo optical imaging.Dendritic polymers or dendrimers present an alternate template for the development of nanoparticulate-based drug delivery and imaging systems. The smaller size (~7–12 nm) of dendrimers have the advantage over the other particles, because its smaller size can possibly improve tumor penetration and the inclusion of tumor specific drug release mechanisms. A Paramagnetic Chemical Exchange Saturation Transfer (PARACEST) MRI contrast agent, Eu-DOTA-Gly4 or a clinical relevant Gd-DOTA was conjugated on the surface of a G5 PAMAM dendrimer. To create a dual mode MRI-optical imaging nanoparticle, Dylight680 was also incorporated on the amines surface of a G5 dendrimer. The particle was detected with  Glioma is a central nervous system neoplasm that typically exhibits hypervascularity, especially grade III and IV tumors with marked heterogeneity. High rate of occurrence and intrusive phenotype leading to poor prognosis make malignant gliomas a major clinical problem. Gliomas represent 78% of all malignant brain tumors, and are the most prevalent cause for cancer-related death in males between age of 20–39 years . InvasivIn clinical practice, T1 relaxation-based contrast agents are being used to discriminate tissue abnormalities in brain as well as other organs . SpecialThere are many ways a relaxation agent can be confined to the vascular bed including conjugation of a simple chelate such as DOTA or DTPA to high molecular weight macromolecules such as albumin, polylysine, or dendrimers of various sizes, prepare large nanoparticles such as iron oxide particles . Many of these larger particles present problems because they tend to be taken up via the liver or spleen. The pore size and the porosity of tumor vessels differ based on the tumor type and the status. Therefore, the effect of nanoparticle-based drug delivery differs based on tumor types, locations, sizes, grades and stages. Similarly, the in vivo effect of dendrimer-based Gd-DTPA contrast agents depends on the dendrimers core, size and the external surface charge ,20. Ther3+ in the nanostructures. Secondly, although T1 weighted images that were collected using dendrimer-based Gd-DTPA, showed a statistically significant contrast enhancement in the glioma tumors, but these results were not confirmed using other imaging methods.This suggests that the pore size in the BBTB of malignant brain tumors has a upper limit of approximately 12 nm ,24,25. Dex vivo and in vivo imaging of the dual-modality dendrimer-based contrast agents showed exquisite possible utility for recognizing the location of gliomas, which demonstrate the benefit of using an exogenous contrast agent for glioma disclosure. Tumor location was identified at a millimeter scale due to the perseverance of the MRI contrast throughout the glioma (Validation of imaging results can perform using dual-modality contrast agents, as the conditions that may affect the analysis of one imaging modality barely affect the other. Thus, these contrast agents can be assembled to take the advantage of two complimentary imaging modalities . MRI is e glioma  and 3 [1ex vivo fluorescence microscopy was used to validate the precision of in vivo MRI for glioma detection. Microscopy images displayed that the contrast agent concentrated in the glioma tumor, but not in the contralateral brain tissue (Furthermore, n tissue . Fluoresn tissue . The mulNonetheless, these successes with lipid-insoluble spherical nanoparticle formulations argues that a combination of targeted therapeutic approaches with nanoparticle formulations can be used to develop “active” dendrimer-based nanoparticles. Keeping this in mind, the surface of dendrimer-based carriers was modified with ligands that can specifically recognize the transferrin receptors that are overexpressed in endothelial cells in brain tumor tissue . This stIn conclusion, a dual mode MRI-optical strategy is a useful method for in vivo biomedical imaging, as noninvasive high resolution anatomical images can be obtained using MRI, while microscopic details in post mortem pathological tissues can be obtained by fluorescence imaging.


Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. Perturbations in DNA synthesis—such as a scarcity of free nucleotides or damaged DNA—leads to replication fork stalling and activation of the checkpoint pathway ,2. The rRecovery from checkpoint activation is key to a successful checkpoint mechanism. During checkpoint recovery, the checkpoint signaling pathway is inactivated, and cell cycle progression is resumed. As the S-phase checkpoint is sensitive to perturbations even under favorable conditions, it is likely that recovery from checkpoint initiation is critical in each and every cell division. It is clear that failure to activate the S-phase checkpoint has deleterious consequences. Stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, leading to genomic instability. Similarly, if checkpoint recovery mechanisms fail, stalled forks can persist, increasing the likelihood of DNA damage. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA would proceed through the cell cycle, also impacting genomic stability.Initiation of the S-phase checkpoint response is dependent on a signaling cascade that is remarkably conserved in eukaryotes. This review will highlight mechanisms identified in simple eukaryotes such as budding yeast and point out distinctions observed in higher eukaryotes, including humans. Both stalled replication forks and DNA damage are recognized by sensor complexes, which activate a kinase cascade to prevent cell cycle progression . In While substantial progress has been made in identifying factors, pathways, and molecular events central to checkpoint activation, our understanding of checkpoint recovery is much more limited in comparison. Recovery from replication stress occurs after the original damage or defect is repaired, thus triggering checkpoint inactivation and a return to progression through the cell cycle ,29.Checkpoint initiation programs must be counteracted to achieve recovery and re-entry into the cell cycle. S-phase checkpoint recovery has to accomplish two key steps: inactivation of checkpoint signaling and resumption of DNA replication.One straightforward way to turn off a signaling pathway is by inhibiting key enzymatic steps in the pathway. There are S-phase checkpoint recovery mechanisms that involve deactivation of the checkpoint signaling pathway by interfering with Rad53 kinase activity A. DirectCheckpoint signaling may also be affected by alterations of chromatin structure and changes to checkpoint protein complex architecture B. DampenTrCP ubiquitin ligase and the proteasome [Protein degradation of checkpoint adaptor proteins is also important for S-phase checkpoint recovery, although whether this degradation is required solely for checkpoint signaling inactivation is not clear. Degradation of human Claspin  is linked to checkpoint recovery after fork stalling caused by exposure to hydroxyurea, which limits free nucleotides ,44,45. Poteasome ,44,46. Ioteasome ,49,50,51oteasome .Dia2 ubiquitin ligase during recovery from the DNA alkylating agent methyl methanesulfonate (MMS). Both checkpoint-phosphorylated Mrc1 and unmodified Mrc1 are degraded, and a Mrc1 mutant protein that cannot be phosphorylated by checkpoint kinases exhibits partial stabilization. Induced degradation of Mrc1 only during recovery rescues the checkpoint recovery defect in cells lacking SCFDia2 ubiquitin ligase activity, indicating that the predominant role of this complex during S-phase checkpoint recovery is degradation of Mrc1. However, induced degradation of a checkpoint-defective version of Mrc1 during the same time period cannot rescue the recovery defect in these cells, suggesting that removal of checkpoint-activated Mrc1 is key to the recovery process [Mms22 ubiquitin ligase counteracts the replicative function of Mrc1  to also facilitate replication fork restart or repair [In budding yeast, the Claspin ortholog Mrc1 is also targeted for degradation during recovery from the S-phase checkpoint C, indica process . In addir repair .Completion of DNA replication after the activation of checkpoint signaling is critical to successful S-phase checkpoint recovery. During checkpoint activation, some proteins at the fork involved in checkpoint signaling, such as ATR/Mec1 and Claspin/Mrc1, promote replication fork stabilization ,55,56,57A growing number of proteins have been identified to have roles in replication fork restart, and multiple fork restart mechanisms have been proposed. In general, these mechanisms can be divided into two groups: direct restart or a broad group of alternative restart mechanisms that require remodeling or recombination to restore DNA replication. The type of replication stress or DNA lesion that led to the fork stall may influence the type of restart mechanism. For example, recovery of a fork stalled by limiting replication components presents a different challenge than a leading strand lesion that has a led to uncoupling of the leading and lagging strand polymerases.Direct restart of a stable, stalled fork is a straightforward approach to complete DNA synthesis during checkpoint recovery. However, we know very little about mechanistic steps required for initiation of direct restart of a stalled fork (re-priming) in eukaryotic cells. For example, it is not clear if re-priming is linked to inhibition of checkpoint signaling. In primates, the methyltransferase and nuclease protein METNASE (SETMAR) is required for restart of the majority of forks following a hydroxyurea-induced checkpoint ,60. InteAlternative ways to restart DNA replication include a variety of mechanisms that may require remodeling by fork reversal, nucleolytic processing of nascent DNA strands, or recombination mechanisms . ProRecombination-based restart mechanisms are probably most relevant to collapsed forks where the replication machinery has been lost, thus facilitating Holliday junction formation. The recombination factor Rad51  can be recruited to stalled forks ,78,79,80We are just beginning to identify and investigate mechanisms of checkpoint signal inactivation. As these mechanisms become better understood, it will be interesting to determine whether they are coordinated into an overall cellular program that facilitates recovery from the S-phase checkpoint. One intriguing question is whether checkpoint activation in response to distinct damage or replication stress triggers specific signaling inactivation mechanisms. Moreover, are checkpoint recovery mechanisms themselves downstream targets of the initial checkpoint activation? It is tempting to imagine that mechanisms that turn off the signaling pathway are folded into the initial activation as a means of limiting prolonged activation.Many of the proteins required for the DNA replication checkpoint and fork restart during recovery are compromised in human diseases. It is easy to imagine that defects in checkpoint recovery might lead to genome instability, and therefore also contribute to human disease. As checkpoint recovery mechanisms become better understood, we look forward to new information about the role of these pathways in protecting human health.


The small-interfering RNAs (siRNAs) have been employed to knockdown the expression of cancer-associated genes and shown some promise in cancer therapy. However, synthetic siRNA duplexes or plasmid mediated delivery of siRNAs have several problems, such as short half-life, low transfection efficiency and cytotoxicity associated with transfection. Conditionally replicating adenovirus (CRAds) as the delivery vector for short hairpin RNAs (shRNAs) could overcome these limitations and have shown augmented anti-tumor effects in experimental studies and preclinical trials. In this review, we summarize recent progress in the development of CRAds-shRNA for cancer treatment. Combination of CRAds-shRNA with chemotherapeutics, radiation, dendritic cells, monoclonal antibodies and small-molecule inhibitors will be necessary to eradicate cancer cells and cancer stem cells and achieve superior outcomes. The use of CRAd platform for efficient delivery of shRNAs and foreign genes will open a new avenue for cancer therapy. RNA interference (RNAi) is a powerful tool for gene knockdown by sequence-specific post-transcriptional targeting . Many stCRAds are natural or genetically modified viruses that selectively replicate in and kill neoplastic cells while sparing normal cells , 4. NumeTo enhance the specificity and efficacy of CRAds for cancer treatment, oncolytic viruses have been modified to combine oncolytic replication with therapeutic transgenic expression. In this review we summarize recent progress in the utilization of CRAd-shRNA in cancer therapy and discuss novel strategies to develop highly efficient oncolytic Ads.Most adenoviral vectors used in RNAi delivery are derived from human serotypes 2 and 5. Carette et al. were the first to propose using shRNA with oncolytic viruses in cancer gene therapy . This syin vitro and inhibited prostate cancer growth and metastasis in xenograft nude mice [The first strategy of oncolytic viruses armed RNAi involves the use of one shRNA. In the prototype, Carette et al. constructed CRAds encoding shRNAs against firefly luciferase and demonstrated that siRNAs expressed from CRAds could suppress the expression of firefly luciferase. In particular, the efficiency of silencing increased during viral replication . Zhang eude mice . Our resin vivo [Yun et al. developed an E1A/E1B double mutant replicating adenovirus Ad-ΔE1Bmt7 , and conin vivo .The second strategy involves the use of dual or multiple shRNAs in one Ad vector. Jazag et al. were the first to establish the method for simultaneous silencing of multiple targets by shRNA-expressing RNAi plasmid vectors . The mulWe postulate that simultaneous targeting of two or multiple tumor-specific genes using oncolytic adenovirus and siRNA enhances therapeutic efficacy against tumors, but could augment the inherent risks, including immune responses, cytotoxicity, off-targeting and oversaturation of endogenous pathways. Moreover, the optimization of siRNA is needed for stable knockdown of multiple genes. These factors should be taken into account when combinatorial RNAi is selected for gene therapy.in vivo was not as adequate as that of in vitro experiments, particularly at late stage [Recently, cancer gene therapy has focused on utilizing cytokines, tumor suppressor, and apoptosis related genes as therapeutic genes , 39. CRAte stage . The phete stage . Therefoin vivo [The nonspecific native tropism of the adenovirus limits the efforts to target cancer cells specifically, and increases the potential for side effects. One strategy is the use of tumor-specific promoters (TSPs) to selectively drive viral E1 expression to increase tumor specificity. This is typically accomplished by placing viral E1A gene under the control of an exogenous promoter that is active or induced in the particular cancer that is being targeted. The most commonly used promoter that augments virus specificity is hTERT, which is active in 85-90% of tumor tissues and is detectable in the early stages of malignancy . hTERT pin vivo .We constructed oncolytic virus G250-Ki67, and G250 promoter-derived CRAds carrying Ki67-siRNA could efficiently amplify and deliver Ki67-siRNA in renal cancer cells, leading to inhibited proliferation and enhanced apoptosis . A recenThe monotherapy of adenovirus has demonstrated limited efficacy in a clinical setting . ComprehPreclinical and clinical trials support that CRAds gene therapy and chemotherapy have complementary or synergistic effects, leading to better anti-tumor effect than either treatment alone. One effective approach in the treatment of malignant tumors is direct injection of CRAds combined with chemotherapeutic agents. Another means of combined therapy is the use of CRAd-shRNA to knockdown genes associated with chemoresistance in tumor cells.in vivo, but the tumors were not eliminated. In contrast, complete tumor eradication was observed when tumors were co-injected with ZD55-siApollon and 5-FU [in vitro and in vivo [Apollon is a membrane-associated inhibitor of apoptosis and is upregulated in chemoresistant cancer cells . Chu et and 5-FU . Another in vivo .Total radiation dose that can be delivered to cancer is limited by the damage to normal tissues that are irradiated during radiotherapy . RecentlTakashi et al. engineered a replication-deficient adenovirus encoding shRNA targeting DNA-PKcs to treat human HCT116 colon cancer cells, leading to increased radiation sensitivity . When CRFor cancer immunotherapy, different kinds of immune cells have been used including dendritic cells (DCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells –70. DCs pRb/E2F pathway is abnormal in many solid tumors . Yang etDespite the advantages of CRAds for cancer therapy, two important issues remain as the big challenges. First, cancer stem cells (CSCs) are thought to account for tumor relapses. Second, clinical trials using advanced anti-cancer therapies fail to reach the outcomes of preclinical studies due to limited distribution throughout solid tumors and quick degradation of the therapeutic payload . TherefoPierce speculated that tumors contain a small number of malignant stem cells that maintain stem cell function and give rise to progenitor cells with various degree of differentiation . Current+CD24low/− breast cancer CSCs in vitro and in vivo [+CD24low/− cells were injected into the fat pads of SCID mice, tumors formed rapidly, but CD44+/CD24−/low cells infected with Ad5/3-Δ24 showed slower tumor formation [A number of studies have explored the use of oncolytic Ads for CSCs targeting. Ad5/3-Δ24 and Ad5.pK7-Δ24 had a 24-base pair deletion in the viral E1A gene, which disrupted the Rb-binding capacity of E1A protein and thereby conferred conditional replication only in cells deficient in Rb/p16 pathway , 84. The in vivo . When CDormation .in vitro and in vivo [Oncolytic adenovirus Delta24-RGD represents a promising therapeutic agent for malignant glioma. Delta24-RGD showed efficacy against glioma  in vivo , and pha in vivo . Histone in vivo .Moreover, transcriptional targeting mediated by adenoviral vectors using promoters active in CSCs may be a powerful approach for the eradication of CSCs . With thin vivo delivery of oncolytic viruses is using cell carriers that chaperone viral delivery to the tumor nodules. Carrier cells for oncolytic virus delivery could shield the virus from host defenses and direct them toward tumors. Mesenchymal stromal cells (MSCs) are interesting candidates as carrier cells, because they are easily obtained, cultivated and propagated in vitro, and can home to tumor microenvironments [The systemic administration of oncolytic virus is often inefficient, because the virus is susceptible to neutralization by blood components, especially antibodies, and sequestration to healthy organs . An alteronments . Using MMSCs have been employed to deliver oncolytic virus which subsequently infect and replicate within malignant cells and eradicate the tumors –97. WhenCRAds can be used as gene delivery vehicles to tumors, in addition they independently induce oncolysis and avoid the damage of adjacent normal cells. A variety of strategies have been designed to enhance the oncolytic effects of CRAds. Among them, two major strategies of CRAds armed with shRNA have been pursued: i) improve the efficiency and selectivity by silencing multiple target genes, co-expression of tumor suppressor genes or using tumor specific promoters; ii) improve CRAds delivery and reduce virus clearance by using MSCs as cell delivery vehicles. Combination therapy with chemotherapeutics, radiation, dendritic cells, monoclonal antibodies, small-molecule inhibitors will be necessary to eliminate cancer cells and CSCs to achieve superior outcomes. The use of CRAd platform for shRNA and transgenic delivery will open a new avenue for cancer therapy.


Coronary revascularization remains the standard treatment for obstructive coronary artery disease and can be accomplished by either percutaneous coronary intervention (PCI) or coronary artery bypass graft surgery. Considerable advances have rendered PCI the most common form of revascularization and improved clinical outcomes. However, numerous challenges to modern PCI remain, namely, in-stent restenosis and stent thrombosis, underscoring the importance of understanding the vessel wall response to injury to identify targets for intervention. Among recent promising discoveries, endothelial progenitor cells (EPCs) have garnered considerable interest given an increasing appreciation of their role in vascular homeostasis and their ability to promote vascular repair after stent placement. Circulating EPC numbers have been inversely correlated with cardiovascular risk, while administration of EPCs in humans has demonstrated improved clinical outcomes. Despite these encouraging results, however, advancing EPCs as a therapeutic modality has been hampered by a fundamental roadblock: what constitutes an EPC? We review current definitions and sources of EPCs as well as the proposed mechanisms of EPC-mediated vascular repair. Additionally, we discuss the current state of EPCs as therapeutic agents, focusing on endogenous augmentation and transplantation. Coronary artery disease (CAD) remains a leading cause of morbidity and mortality . CoronarThe vessel wall's response to injury  depends on many interdependent factors, including the insult itself in combination with the dynamic nature of the vessel wall-stent interface , vascular tone, and circulating signals. Indeed, PCI itself prompts endothelial denudation and disruption of vascular homeostasis given the high pressure balloon inflation and subsequent force transmitted by stent struts to the endothelial layer extending damage through the underlying medial and adventitial layers . In respEPCs represent an attractive therapeutic option given their purported ability to promote vascular repair after stent placement. Accordingly, an inverse correlation between circulating EPC numbers and both atherosclerotic burden and probability of future cardiovascular risk has been noted , 15. FurVascular progenitor cells were first observed in animal models when implanting Dacron grafts or silastic tubes –29. ThesSince their discovery, the precise definition of EPCs remains the subject of considerable debate and represents a major hindrance in understanding the biology and application of these unique cells. This is believed to be a driving force behind conflicting results noted in both preclinical and clinical trials, where differing methodologies in cell sources, cell purification, detection methods, and animal models or assays have been published . Thus faEPCs can be classified based on the expression of specific cell-surface antigens via flow cytometry . Mature EPCs can also be isolated from peripheral blood by plating these cells in various cell culture assays and assessing their colony forming ability . Asahara+/VEGFR2+/CD133+ cells were isolated using a cell sorter and assayed using two different endothelial culture systems and hematopoietic colony assays. No endothelial colonies formed and the isolated cells expressed the hematopoietic cell marker, CD45 [Identifying the relationship between these two different isolation methods for EPCs is vital to ensuring a uniform definition of EPCs. One study examined the relationship of both methods and revealed that CFU numbers did not correlate to numbers of either CD34+/KDR+ or CD34+/KDR+/CD133+ cells . In 2007er, CD45 . One poser, CD45 . Hence, β-galactosidase), which was transcriptionally regulated by an endothelial-specific promoter (Tie2 or Flk-1). The group identified LacZ-positive cells in the developing neovasculature in all the models [Asahara et al. described the first EPCs as originating in the bone marrow . The bone models . Since te models , 49. More models .Renal progenitor cells (RPCs) were isolated from normal adult human kidney cortical parenchyma with cell-surface markers CD133+ and PAX-2+ . These RPCs were capable of self-replication and could be differentiated in vitro into epithelial or endothelial cells . “NephroMost models of EPC-mediated vascular repair invoke the homing capability of bone marrow-derived EPCs that are circulating in the blood. It is believed that human aortic endothelial cells (HAECs) or human umbilical vein endothelial cells (HUVECs) from vessel walls are terminally differentiated into mature ECs . However−/− mice were used to identify adventitial progenitor cells stained by Sca-1, CD34, and Flk-1 [Recently, in addition to circulating progenitor cells, the existence of resident vascular progenitor cells within the adventitia of blood vessels has been demonstrated. ApoEnd Flk-1 . Indeed,nd Flk-1 . In 2007nd Flk-1 . They isnd Flk-1 .Adipose tissue was identified as a source of EPCs when Planat-Benard et al. isolated human adipocytes and dedifferentiated them into cells capable of then differentiating into adipocytes or ECs under appropriate conditions . IsolatiSeveral studies have confirmed an important role for the spleen in EPC mobilization and transplantation . Most heThe predominance of evidence supports bone marrow-derived EPCs as the most likely source for arterial repair and accordingly we will focus on their mobilization, reviewing various chemokines responsible for guiding EPCs to sites of vascular injury. VEGF, an effective mobilizer and activator of angiogenesis, is believed to induce proliferation, differentiation, and chemotaxis of ECs following vessel injury . AccordiSecondarily, SDF-1 is a chemokine of growing interest, a constitutively expressed protein that is upregulated by inflammatory mediators, extracellular matrix changes, mechanical forces, and hypoxia . Plateleβ2-Integrins on the surface of EPCs mediate migration and adhesion of EPCs to the damaged endothelium as well as neovascularization [β1- and β2-integrins on the surface of EPCs by binding to HMGB1 receptors RAGE (receptor for advanced glycation endproducts) and TLR2 (toll-like receptor 2). Interestingly, HMGB1 is released into the extracellular space upon necrosis, but not apoptosis, and was associated with improved homing and adhesion of EPCs to the site of injury [Understanding the mechanism by which EPCs home to sites of vascular injury is critical to harnessing their therapeutic potential. This process is thought to be similar to the rolling and adhesion behaviour exhibited by leukocytes in the setting of inflammation. EPC homing involves an interaction between EPC surface molecules and associated ligands expressed on dying ECs. P- and E-selectin are believed to play critical roles in this process. Foubert et al. have shed light on this process through their studies on the erythropoietin-producing human hepatocellular carcinoma (Eph) receptor and its associated ephrin ligands, which are regulators of vascular development. The group has shown that Eph4B activation by ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs in a hind limb ischemia mouse model and that this response is blunted with EphB4 siRNA treatment. Eph4B appears to elevate P-selectin glycoprotein ligand-1 expression and EPC adhesion, an effect that can be abrogated with utilizing neutralizing antibodies to E- and P-selectin . β2-Interization . High mof injury .Next, intercellular adhesion molecule-1 (ICAM-1) upregulation in ischemia has been noted to increase EPC recruitment to ischemic limbs . As a hyApart from EPC migration to sites of vascular injury, EPC invasion of injured tissue is critical for organ repair and function. Gene expression profiling of EPCs and mature ECs has identified the protease Cathepsin L (CathL) as being highly expressed. CathL was shown in vitro to be crucial for matrix degradation and EPC invasion. A CathL-deficient hind limb ischemia mouse model characteristically shows poor limb recovery. As well, when CathL-deficient progenitor cells are infused, impaired homing to sites of injury and impaired neovascularization are observed . EPCs laIt is unlikely that EPC transdifferentiation into mature ECs represents a meaningful reparative mechanism. Rather, an increasing number of studies have shown that these circulating cells may facilitate arterial repair through paracrine influence on neighbouring cells . Many ofParacrine signaling appears to be an important aspect in both early and late EPCs. Early EPCs are derived from a monocytic cell lineage (CD14+) to form spindle-shaped cells after 7 days of culture. In contrast, late EPCs are derived from a CD34+ lineage after 14–28 days in culture and resemble ECs . Early EMore recently, EC-derived microparticles (EMPs) or microvesicles (MV) have been identified as vehicles for different paracrine modes of action. EMPs are complex vesicles shed from activated or apoptotic ECs which play a major role in endothelial function and angiogenesis. EMPs are highly functional molecules with several surface molecules and contain DNA, RNA, or microRNA . These EPreclinical trials involving EPCs have shown promising results, with EPCs demonstrating the ability to specifically incorporate into the site of injury and mitigate neointima formation . Our groSome common antihypertensive  agents include angiotensin converting enzyme inhibitors (ACEi), angiotensin II receptor blockers (ARB), and calcium channel blockers (CCB). All of these agents have been extensively studied and yielded an improvement in EPC number and function by differing mechanisms of action. ARBs have improved numbers and function of EPCs by inhibiting oxidative stress , while AStatins were originally developed to modify lipid profiles in patients by reducing LDL and triglyceride levels to improve cardiovascular outcomes. However, clinical studies involving statin therapy in patients with CAD showed higher levels and mobilization of EPCs , 121. Thβ) is a serine/threonine kinase that phosphorylates β-catenin to negatively regulate the Wnt signaling pathway [β was associated with increase in EPC levels and reduction of EPC apoptosis. In vivo, we observed that GSK-3β inhibition resulted in an improvement in reendothelialization and reduction of neointima formation following injury [Glycogen synthase kinase  have reduced EPC levels and increased rates of apoptosis. In addition, diabetics have shown higher level of GSK-3f injury . Our grohibition . Althoughepsin B . Althoug1 to S phase progression in cell cycle via inhibition of mammalian target of rapamycin (mTOR) kinase [As discussed, coronary stents have markedly advanced from simple metal scaffolds to sophisticated drug-delivery systems to facilitate arterial healing . Drug-el) kinase . As the ) kinase .The first stent to harness the healing capacity of EPCs was the Genous monoclonal anti-human CD34 coated stent . This EPTo further refine this technology, the EPC-capture stent has been combined with a paclitaxel-coated balloon with intent of reducing the noted NI proliferation and ISR that plagued first-generation Genous stents given its antiproliferative properties. The Perfect Stent study studied 120 patients with CAD who were treated with EPC-capture stent followed by paclitaxel-eluting balloon after dilatation. This approach was successful in reducing restenosis  and may Stem cell therapies have been developed as new treatment regimen for patients suffering from acute myocardial infarctions since the early 2000s. The first of these trials involved the injection of bone marrow-derived mononuclear cells (BMMNC) in 10 patients at the site of infarct following balloon dilatation . Since t6 to 1010 cells [The procedure involves harvesting bone marrow under general anesthesia from the bony pelvis. CD34+ or CD133+ hematopoietic progenitor cells (similar surface markers as the EPCs defined earlier) are isolated from the BMMNC and then cultured for 2–4 weeks to obtain a sufficient yield of progenitor cells. These cells are then injected directly into the patient's heart at the site of injury during PCI. The timing of stem cell injection varies in studies from first 24–48 hours to four weeks after PCI. Also, the dose of cells that were administered varied considerably within these large trials anywhere from 1010 cells –149. Ove10 cells . While cOur understanding of the complex environment that is the vessel wall remains incomplete. Certainly, the discovery of EPCs holds great potential for both the monitoring and therapy of vascular disease, particularly in repairing the endothelial injury incurred by modern interventions. Be it by augmentation and exploitation of endogenous cells or direct transplantation of cells to areas of need, both approaches show promise. However, despite considerable efforts over the past few decades, our understanding of the definition, signaling, and differentiation of these enigmatic progenitor cells remains imperfect, hampering further developments. Only once these fundamental components are clarified will progenitor cells be able to advance and emerge as a legitimate therapeutic option.