A functional vascular system is indispensable for drug delivery and fundamental for responsiveness of the tumour microenvironment to such medication. At the same time, the progression of a tumour is defined by the interactions of the cancer cells with their surrounding environment, including neovessels, and the vascular network continues to be the major route for the dissemination of tumour cells in cancer, facilitating metastasis. So how can this apparent conflict be reconciled? Vessel normalisation—in which redundant structures are pruned and the abnormal vasculature is stabilised and remodelled—is generally considered to be beneficial in the course of anti-cancer treatments. A causality between normalised vasculature and improved response to medication and treatment is observed. For this reason, it is important to discern the consequence of vessel normalisation on the tumour microenvironment and to modulate the vasculature advantageously. This article will highlight the challenges of controlled neovascular remodelling and outline how vascular normalisation can shape disease management. In early development, vasculogenesis involves the de novo formation of blood vessels by endothelial precursor cells (EPCs), which follow cues from growth factors and cytokines to create a primitive vascular tree; this vascular tree then gets remodelled and expanded by another mechanism, angiogenesis.14 some indication can be gained by indirectly assessing the number of cells that circulate in the blood.15In healthy adults, endothelial cells tend to be quiescent and their turnover is very low compared with cells from other organs, for example, the gut. With the exception of the female reproductive cycle, angiogenesis is almost always pathological and the result of a trauma, surgery or an illness, such as cancer. Cancer cells are able to grow into solid tumours only by procuring a supply of nutrients and oxygen to meet their increasing demands for energy. They achieve this by creating a blood supply, either by exploiting the existing vasculature through co-option or vascular mimicry and/or by coaxing the existing vessels to expand through angiogenesis. As mentioned before, bone marrow-derived EPCs are also able to contribute to new vessel formation in an adult, although it is difficult to determine the exact extent of their contribution towards the tumour vasculature;16 This independence of growth factors can be a bypass around typical modes of anti-angiogenic targeting before sprouting angiogenesis prevails again.18The majority of solid tumours are sustained by sprouting angiogenesis, in which endothelial cells can become tip cells or stalk cells Fig. . Not all20 Evidence that existing host vessels are indeed seeding new vasculature following an activation signal and begin to sprout emerged with Judah Folkman’s ground breaking discovery in 1971 of a ‘tumour angiogenesis factor’.21 The theory had been that if a cancer could be stopped from growing its own blood supply it would wither and die. We now know that a ‘tumour angiogenesis factor’ does not exist in this simplistic form. Rather, a plethora of growth factors, together with other cytokines and biomolecules of various shapes and sizes, are involved in the process of angiogenesis (even different mechanisms of angiogenesis itself),25 such that targeting only one of the factors or receptors often spontaneously leads to the upregulation or activation of another factor to compensate for the loss.26 We also know that an intact, functional vascular system constitutes a key systemic route for the delivery of drugs and in particular is required for the responsiveness of the tissue microenvironment to these, and other, therapeutic agents, especially in the case of the tumour microenvironment (TME). Biologically and mechanistically treatment resistance is best understood in the context of tumour hypoxia. In the absence of a functional vascular network oxygenation of the TME is insufficient hence rendering chemotherapy, radiation and photodynamic therapy less efficient and the success of the latter depends on the generation of reactive oxygen species (ROS).28The fact that the circulation is required for tumour growth was known before the expression ‘tumour angiogenesis’ was coined.Thus, targeting the blood supply of a tumour presents a challenge Box . What haBox 1Endothelial cell heterogeneity146 Endothelial cells are capable of sensing and responding to their environment, which accounts for their phenotypic heterogeneity. Separated from their vascular bed and cultured in situ, they undergo a phenotypic drift. DNA microarrays reveal that about 50% of their site-specific genes are lost after passage. Concurrently, some specific properties are retained under cell culture conditions, which means that their gene expression is mediated by both their immediate environment and by epigenetics.147The vascular system can be classified according to size, function, cell morphology, gene and protein expression, depending on the organs that are supplied—in other words, on the nature of the vascular bed. Arteries, veins, arterioles and venules are larger vessels that narrow into a network of capillaries. The larger vessels consist of a continuous endothelium, held together by tight junctions, with three strong outer support layers. Capillaries are fine and narrow vessels that can be continuous, fenestrated or discontinuous, and are stabilised by the basal lamina and pericytes. The phenotypes of endothelial cells vary with their function and vascular bed. Extreme examples would be the highly fenestrated or discontinuous endothelium of capillaries in filtrating organs, like the kidneys and liver, and the tight endothelium of capillaries in the brain that constitutes the blood–brain barrier.Endothelial cell heterogeneity in tumour vesselsThe phenotype of tumour endothelial cells (TEC) differs greatly from the normal endothelial cells in the host organ. The epigenetic footprint of endothelial cells in the tumour vasculature, however, is conserved, meaning the specific properties linked to their original vascular bed are kept.148For a long time, it was accepted that TEC cells within tumours were genotypically equal to their normal counterparts and that any changes in phenotype were a mere reaction to the cancerous environment. Today it is appreciated that TECs can display cytogenetic abnormalities. TECs derived from malignant melanoma and liposarcoma are not normal diploid cells but contain an abnormal number of chromosomes in the nucleus (aneuploidy). The chromosomal instabilities are not clonal but heterogeneic within a TEC population. However, the mechanisms that cause TEC heterogeneity are still pinned on the tumour environment.30 A relentless exposure to angiogenic factors released from the hypoxic tumour environment renders the endothelial cells in a state of constant activation, resulting in an uneven and dilated lumen and a tortuous vessel architecture  from being directional (movement from a high-pressure region to a low-pressure region) to being non-directional (‘diffusion’). Diffusion through the dense extracellular matrix (ECM) is extremely slow so that some macromolecules can be re-absorbed into the circulation. This has been exemplified experimentally for globular IgG, albumin and dextrans using fluorescence correlation spectroscopy.37Whichever of the mechanisms that a solid tumour uses for increasing the blood supply , the newly formed vessels (neovessels) are abnormal in the majority of cases.ure Fig. . Despite38 The drug was discovered through random screening and selected for its cytotoxic properties. At the time, it was largely unknown how ICRF-159 changed the architecture of the tumour vessels. Today we know that ICRF-159 is an inhibitor of the platelet-derived growth factor-β (PDGF-β) receptor, and its anti-angiogenic properties and potential as a vessel normalisation drug have been established.39 The response of the pathological neovasculature to anti-angiogenic treatment had not been anticipated and the effect that normalisation has on malignant and non-malignant cells opened new avenues for cancer combination therapy.42The rationale behind anti-angiogenic treatment is that blocking blood vessel formation in tumours or its regression will deprive cancer cells of nutrients and oxygen and finally starve tumours to death or induce tumour dormancy. However, anti-angiogenic treatment fails to bring about permanent de-vascularisation; rather, normalised tumour vessels emerge from the neovascular tree. Drug-induced vessel normalisation was observed and documented in 1972 by Le Serve and Hellman, when treatment with ICRF-159 (razoxane) was seen to cause the chaotic tumour neovasculature of Lewis Lung Cancer (LLC) in mice to appear normal.43 The immature parts of the neovasculature seem to be more susceptible to anti-angiogenic treatment than more mature vessels are, indicating that mature vessels are evidently less dependent on angiogenic factors. Tumour vessels that do not regress after anti-angiogenic treatment appear normalised, as illustrated in Fig. 44 However, the most important biological aspect of normalised vessels is that a reliable circulation replaces the previously flawed system, such that restored perfusion reverses almost all the abnormalities.Vessel normalisation almost always follows the same recognisable pattern in terms of vascular structure plasticity and changes to the TME.46 Contemporary image analysis software allows for the quantitative analysis of vessel branching, number of sprouting endothelial cells, vessel length and diameter. Spatial image analysis of distance, for example, between pericytes and the endothelium is an indicator of vessel maturity. Blood flow and perfusion can be monitored by systemic optical tracers or non-invasively by functional magnetic resonance imaging (fMRI) and photoacoustic imaging.48 Visual and functional assessment of vascular normalisation is usually supported by complementary analysis at the molecular level, such as the analysis of the expression of angiogenic factors.In animal models, vessel normalisation is mostly observed by microscope image analysis, either at fixed time points or in real time .49 In reality, a combination of these restored normal blood vessel characteristics usually appears, with degrees of intra and inter tumour variation, which might depend on the tumour type and treatment.We can define normalised vessels according to several prominent characteristics. A significant drop in angiogenic factors results in fewer sprouting endothelial cells. Mural cells, such as pericytes, adhere again to the vessel walls and pericyte coverage increases. Through remodelling and reduced branching, leaner and less tortuous vessels with an even luminal diameter emerge, reducing the blood back flow that is typical for abnormal vessels and restoring a uniform blood pressure. Pruning of truncated vessels and endothelial structures that lack a lumen improves the perfusion. In the surrounding tissue, oxygen levels rise and hypoxia decreases, which, in turn, reduces hypoxia-induced activation of angiogenesis. In short, blood vessel maturation takes place.The signalling pathways that lead to sustained angiogenesis are illustrated in Fig. 39 Early research to prove the hypothesis of vessel normalisation used DC101, a monoclonal antibody against VEGFR-2. A single intravenous dose of DC101 administered to tumour-bearing mice could reduce the interstitial fluid pressure by 50% without evidence of lymphatic vessel restoration.46 Extensive vascular pruning of vessels in the tumour was observed through a dorsal window chamber. Vascular density dropped and vessel diameter shrunk. In addition, metalloproteinases that degraded layers of the basement membrane to a normal thickness were activated, and levels of angiopoietin-1 (Ang-1), which supports the recruitment of new pericytes, increased temporarily. Another important finding from these investigations was that the vascular normalisation effect was transient and occurred 3–5 days post treatment, and that, during this time, the response to radiation therapy was greatly improved.51 These findings gave rise to the idea that, rather than inhibiting angiogenesis altogether, controlling angiogenesis would be a preferable approach.As represented in the table in Fig. 52 and increased oxygen levels and reduced hypoxia suggested normalisation of the vessels. Despite controversy about the biological properties and function of VEGFxxx isoforms they were reported to promote angiogenesis while VEGFxxxb isoforms  have been shown to inhibit angiogenesis.53 Re-establishing the natural equilibrium of angiogenic and anti-angiogenic VEGF-A splice variants might offer an alternative means of tuning the angiogenic switch instead of blocking it. Although this method of regulation was achieved by locally adding the anti-angiogenic variant, modulation on a transcriptional level by inhibiting SRPK-1, the key factor in the spliceosome that regulates VEGF-165/VEGF-165b expression might be a more promising approach in the future.56 The regulatory effect of the VEGF-165b variant on the neovasculature is considered particularly advantageous in diabetic retinopathy, where a normalised vascular network is preferable over a significant de-vascularisation.57VEGF is highly expressed in hypoxic areas. The ectopic expression of a recombinant soluble form of VEGFR-2 in melanoma cells under hypoxic conditions did indeed bind murine and human VEGF sufficiently to slow down endothelial cell growth in vitro and reduce the tumour mass in vivo,59 Blocking TGF-β and its receptors decreased the tumour size and prevented metastasis in breast cancer and glioblastoma models and significantly improved the intratumoural penetration of low-molecular weight chemotherapy drugs and nanoparticles in breast cancer models. The effect was attributed to tumour vessel normalisation, as a higher number of perfused vessels was counted and pericyte recruitment to these vessels was observed.Angiogenesis is regulated by a complex network of direct and indirect factors. In many ways, these factors are not entirely independent of VEGF/VEGFR-2 signalling. Some of them bear, for example, a resemblance to VEGFR because they are also tyrosine kinase receptors and signal via the same pathways, and/or trigger VEGF upregulation or otherwise influence the VEGF/VEGFR-2 pathway further downstream. Although it mediates downstream signalling by inducing serine/threonine kinase activity, rather than by activating a tyrosine kinase receptor, transforming growth factor β (TGF-β) plays a role in tumour progression.61Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor that, among several other functions, activates H-Ras to stimulate angiogenesis, and phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt), which both regulate angiogenesis downstream of VEGFR-2, are additional targets that could be considered to achieve vascular normalisation. Selectively blocking one or more of these molecules resulted in persistent vascular changes for up to 2 weeks post treatment in human xenograft-bearing mice.62 Although transcriptomic profiles can vary between tumour vessels and normal vessels, few genes directly associated with the abnormal neovasculature have been identified.59 The regulator of G-protein signalling 5 (Rgs5) has been shown to be a master gene for abnormal vascular morphology in the tumour.63 Rgs5 is expressed in a variety of organs and upregulated on blood vessels in Rip1-Tag5 tumours. Pericytes around the vessels of these tumours were predominantly PDGFR-β positive. Crossing Rip1-Tag5 with Rgs5−/− mice resulted in vascular remodelling and pericyte maturation, which was assessed by α-smooth muscle actin (αSMA) and neural/glial antigen 2 (NG2) expression. Additional observations such as reduced vascular leakage, increased perfusion and higher oxygen levels are consistent with vascular normalisation. Other tumour models crossed with the Rgs5−/− mice also generated similar findings. A gene whose expression directly relates to abnormal vessel formation could theoretically become a target for therapeutic genome editing.64Endothelial cells in abnormal tumour vessels are not homogeneous, and differ in phenotype from their non-tumour counterparts.61As well as blocking factors that are actively proangiogenic, promoting factors that initiate vascular maturation and sustain maintenance were found to normalise the vessels in a tumour. Unlike oncogenic H-Ras GTPases, R-Ras has been identified as an inhibitor of endothelial cell proliferation and functions instead as a regulator of vessel integrity and maturation during vascular remodelling to promote normalisation of tumour vasculature.66Members of the family of angiopoietins have contrasting effects on the vessels. Ang-1 promotes vessel maturation and pericyte recruitment, whereas Ang-2 promotes endothelial cell death and vascular disruption and also works as an antagonist to Ang-1 via the Tie-2 receptor. Inhibiting Ang-2 while simultaneously activating the Tie-2 receptor leads to vascular normalisation in glioma and LLC in mouse models.67 The class 3 semaphorins Sema3A and Sema3F are assigned to the vascular system and counterbalance the actions of VEGF via the Sema3A–neuropilin receptor (NRP-1) complex. In tumour angiogenesis this balance falls in favour of VEGF, but in studies that followed the targeted delivery of Sema3A, tumour angiogenesis was inhibited and vessels regained a functional morphology.69Semaphorins comprise a large and diverse family of secreted, membrane-associated or membrane-bound signalling proteins that are essential for the development and maintenance of many tissues.70Nucleolin is predominantly localised in the nucleolus but is also found on the surface of proliferating endothelial cells. Its inhibition using a blocking antibody reduced tube formation and led to vessel normalisation in vivo, possibly via endothelial cell apoptosis as the levels of the anti-apoptotic molecule Bcl-2 were also reduced.71 In sprouting angiogenesis, Notch-1/DLL4 signalling shapes the vascular network by downregulating the VEGF-induced activation required for an endothelial cell to become a tip cell, which leads the way for proliferating stalk cells that eventually form the tubular network. Notch-1-deleted endothelial cells therefore preferentially become tip cells, which promotes an abnormal vasculature.73 The anti-malaria drug chloroquine was found to induce vessel normalisation by stimulating the Notch-1/DLL4 signalling pathway.75 In vivo, chloroquine reduced the tumour mass and improved the tumour milieu by increasing tissue perfusion, thereby lowering hypoxia and reducing tumour cell invasion while enhancing tissue sensitivity to chemotherapy.Notch/Delta like ligand (DLL)1 signalling is an important determinant of cell fate in early development.76 as the normalisation of TEC metabolism is a major contributor to tumour vessel normalisation. Notably, pharmacological inhibition of tumour-cell-specific cyclooxygenase (COX)-2 leads to reduced expression of VEGF and, consequently, of PFKFB3, the gene that encodes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, in the TECs. Treatment with a COX-2 inhibitor normalises glucose metabolism in TECs and inhibits PFKFB3-mediated endothelial cell motility, leading to a reduction in the formation of tip cells and filopodia and decreased branching in the neovessels.77 An earlier extensive study that used either a blocking agent against PFKFB3 or transgenic mice +/− knockout mice, with the aim of slowing glucose metabolism, resulted in a similar normalisation effect on the neovessels.78The TME in solid cancers is acidic as a consequence of hypoxia and increased glucose metabolism. The metabolism of tumour endothelial cells (TECs), which are also hyperglycolytic, has gained attention over past years,79 However, fractionated radiation, in which the dose is split and given over several intervals allows time for the normal cells to recover and undergo repair as shown in an orthotopic prostate xenograft mouse model of PC3-luc cells; here, the mice were treated with fractionated radiation of 2 Gy on each of 5 consecutive days over a total time of 2 weeks. Examination of the tissues at given time points  revealed a remodelled vascular network with increased pericyte coverage and positive normalisation effects, such as increased perfusion and low hypoxia.80Tumour blood vessel normalisation can also occur without specifically targeting blocking or activating molecular angiogenic signalling factors. Cell death in response to radiation is largely dependent on the dosing and time frame of the therapy. A single high dose of radiation (30–60 Gy) is known to destroy most cells in the targeted tissue, causing a lasting decrease in vascularity.82 When the underlying mechanisms of the influence of moderate exercise on tumour vessel normalisation were studied in detail in vivo and in vitro,83 the increased blood flow after exercise was seen to confer mechanical force on the endothelial cells. The resulting fluid shear stress on the vessel walls stimulates endothelial cells, triggering the production of nitric oxide and vascular remodelling mechanisms.84 The neovasculature of exercised tumour-bearing mice demonstrated all the signs of vessel normalisation, except for pericyte coverage. When the authors further investigated the effect of shear stress on endothelial cells, they observed that serum from exercised mice inhibited the formation of typical vascular structures by endothelial cells cultured on matrigel, suggesting that soluble angiogenesis inhibitory factors were secreted in the mouse model.85 Experiments on endothelial cells cultured under shear stress confirmed this observation. Subsequently, the authors discovered that the activation of nuclear factor of activated T cells (NFAT), via calcineurin, in endothelial cells is central to vascular remodelling in response to shear stress and leads to the upregulation of thrombospondin-1 (TSP-1), an inhibitor of angiogenesis. TSP-1 is essential for exercise-induced vessel normalisation.85Compelling observational evidence indicates that regular exercise synergises with anti-cancer therapy and rehabilitation. Data from preclinical studies suggest that physical activities/exercise promote tumour vessel maturity, followed by positive changes in tissue and oxygen perfusion and drug delivery and a boost to the immune response, all of which combine to improve the outcome of chemotherapy and radiotherapy in individual patients.86 It was originally thought that this outcome occurred because tumour cells, once starved and weakened, were more susceptible to treatment. This turned out to be only a partial explanation, because less perfused tissue tends to be hypoxic, and hypoxic tissue is ultimately more resistant to radiotherapy and chemotherapy.27 The main explanation for the benefit on patients was that anti-angiogenic treatment promoted vessel normalisation. Improved perfusion is overcoming physiological barriers to tissue oxygenation and makes radiation more effective owing to a decrease in hypoxia-induced radiation resistance.28 Reducing vessel leakiness decreases the interstitial hypertension and restoring a distinct pressure gradient leads to the deeper penetration of macromolecules (chemo-therapeutics) in the tissue and facilitates the migration of immune cells.Even before systematic research into vessel normalisation began, the benefit of anti-angiogenic treatment in combination with chemotherapy and/or radiation became obvious. Patients who received bevacizumab, a humanised blocking antibody to VEGF-A, in combination with chemotherapy in clinical trials fared much better than patients who were given either treatment alone.87 In its early stages, a tumour engages in crosstalk with the innate immune system, and innate immune cells, such as macrophages, monocytes and dendritic cells that patrol the blood, accumulate at the neoplastic site. So why can the immune system not prevent tumour growth? In cancer, pathogen recognition triggers an acute inflammatory response, which recruits cytotoxic immune cells. These cells recognise and eliminate the more immunogenic cells, but this process selects for less immunogenic, often more aggressive, cells. As part of the TME, endothelial cells actively participate in the attraction of immunoregulatory factors, and can enhance or suppress the immune response, depending on their interactions and their expression of inflammatory cytokines.88 Lymphocyte trafficking, for example, is highly orchestrated: many molecular and physical factors must align to facilitate the initial and crucial step—lymphocyte rolling—in the process that enables T cells to exit the endothelium and make contact with the antigen-presenting cells (APCs).89 Although endothelial cells are not professional APCs, they do possess the capacity to express MHC-I and MHC-II molecules and can act like APCs.91 However, the presence of an aberrant tumour vasculature means that none of these factors can be co-ordinated.94 Consequently, the inflammatory response in a tumour no longer has the positive effect of an acute reaction to harmful stimuli but becomes a chronic inflammatory site; to quote Harold Dvorak, “tumours are wounds that never heal”.96 That may explain why a majority of patients do not benefit from anti-cancer immunotherapy in the long term. Vessel normalisation, linked to increased tissue perfusion and reduction of stromal components such as cancer-associated fibroblasts (CAFs) and collagen, to mention a few, directly promotes immune cell infiltration and functionality and as a result enhances the response to immune therapy.97The immune response to cancer could be considered as a specialised case of immunity.93 and unrestricted blood flow and restored perfusion of the tissue reduce hypoxia. Hypoxia is known to contribute to immune resistance and immune suppression in the TEM,98 as hypoxic regions are highly infiltrated by immune-suppressive cells such as myeloid-derived suppressor cells, tumour-associated macrophages and T-regulatory (Treg) cells, which are immune-suppressive.100 Vessel normalisation also restores the functionality of the luminal lining, or glycocalyx, in the neovessels, which is essential for leukocyte rolling.101 High levels of VEGF suppress T-cell infiltration into tumours by decreasing the expression of the T-cell-attracting chemokines CXCL10 and CXCL11; consequently, this effect is reversed upon vessel normalisation.102As indicated above, blood vessel normalisation leads to an improved immune response, involving T-lymphocyte recruitment from the secondary lymphoid organs to the tumour tissue and the accumulation of functional immune cells in the TEM,104 PD-1, alongside cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3), is a well-studied inhibitory immune checkpoint that negatively regulates T-cell effector function and, by themselves synthesising these checkpoint proteins, cancer cells can avoid being attacked.107 Vascular normalisation mediated by low-dose anti-angiogenic treatment can convert cancers that are unresponsive to immunotherapy into responders, as demonstrated in a small group of patients with glioma in whom the beneficial effects of the immune checkpoint inhibitor ipilimumab were observed only in combination with bevacizumab.108 A combination of bevacizumab and CTLA-4 immune checkpoint inhibitors has proven favourable for the treatment of melanoma patients, possibly by evoking humoral immunity to galectin-1, an angiogenic and pro-tumour factor.110The success of treatment using immune checkpoint inhibitors is increased in patients with a higher number of pre-existing tumour-infiltrating immune cells that express PD-L1, the ligand for programmed death-1 (PD-1).111 Eosinophils contribute to tumour immunity by secreting chemoattractants for CD8+ T cells. The intravenous transfer of activated eosinophils into MO4 (mouse melanoma) bearing mice (in combination with activated T cells) induced a remarkable normalisation effect on the neovasculature, including enhanced pericyte recruitment and downregulation of the Rgs5 gene.112 Activated eosinophils altered the TME by shifting the balance of TAMs from the invasion- and metastasis-promoting phenotype, M2, to the classically activated phenotype, M1.114 A similar effect on macrophage polarisation could be achieved by inhibition of the Ang/Tie2 and the pyruvate dehydrogenase/hypoxia-inducible factor 1-α (PDH/HIF-1-α) pathways.116 CD4+ and CD8+ T cells adoptively transferred into Rgs5-negative Rip1-Tag5 mice prolonged their survival because the T cells were able to significantly infiltrate the ‘normalised’ tumours of the knockout mice but not of wild type mice.63 Experimentally disrupting the vessel normalisation process using an NG2-knockout mouse model (which hampers pericyte recruitment) resulted in reduced CD4+ TH1 cell infiltration. In turn, the depletion or deactivation of CD4+ TH1 cells decreases tumour vessel normalisation.117 T cells are known to secrete interferon γ, which is known to regulate angiogenesis.119 The normalised tumour vasculature and CD4+ TH1 cell immune response create some sort of positive feedback loop that generates a controlled angiogenic response.121It also transpires that an improved immune response is not only a consequence of tumour vessel normalisation but that it also contributes to vessel normalisation. Patients with tumours that were eosinophil-rich had better overall prognoses than those with fewer eosinophils.123 In a subset of patients with newly diagnosed glioblastoma within a Phase 2 trial that had received a regime of cediranib and chemoradiation, better perfusion and, consequently, higher oxygenation of the tumour tissue were associated with improved overall survival. Oedema in the brain was reduced and responses to radiotherapy were enhanced.124In a clinical study in which glioblastoma patients were given a single dose of AZD2171  signs of vessel normalisation—based on the calculation of a ‘vascular normalisation index’—lasted for as long as 28 days, with some features of the normalised network persisting for up to 4 months. Vascular permeability/flow and microvessel volume assessed by fMRI correlated positively with the clinical outcome of patients with glioblastoma. For most patients who showed a high degree of vascular normalisation after a single dose of cediranib only, the clinical outcome improved.125 Although the anti-angiogenic treatment of brain tumours and accompanying vessel normalisation are associated with an overall benefit in some studies, data from other studies have revealed conflicting results. Keunen et al. reported that treatment of human glioblastoma xenografts in rats with bevacizumab decreased the number of larger sized vessels and rendered the tumour tissue more homogeneous but also increased tumour cell migration into healthy parts of the rat brain.126 The authors suggested that a drop in tumour oxygenation caused by fewer blood vessels triggered a metabolic change in the tumour cells that shifted them towards an invasive phenotype. Whether or not these findings translate to end-stage glioblastoma patients treated with bevacizumab is unknown.127The dose-dependent response to bevacizumab in the orthotopic glioma mouse model U87 showed that vessel regression and vessel normalisation occurs in animal treated with low  and medium to high doses. Tumour regression and prolonged survival of the animals, however, was only observed in the medium to high dose group. The authors did not find bevacizumab triggered invasive behaviour of the glioma cells within their treatment time frame of 12–25 days.129 Furthermore, gliomas are amongst a special group of cancers that are known to use vascular co-option for the growth of primary tumour sites and for metastasis. The cancer cell cytoskeleton and cell motility are fundamental factors in the molecular mechanisms of vascular co-option and mediated by Cdc42, a small Rho GTPase that regulates filopodia formation of the actin cytoskeleton and integrins.132 Integrin adhesion via the β1 chain seems essential for cancer cells to adhere to endothelial cells to both invade and escape from the vascular system through intercalation.104 In the brain, glioblastoma cells with cytoskeletal actin extensions, called flectopodia, adhere to the blood vessels by connecting in an astrocyte-like manner to the supporting pericytes suggesting that vessel normalisation and stabilisation by pericytes could potentially support vascular co-option and hence metastasis.134 However, a strong link between vascular co-option and vessel normalisation after anti-angiogenic treatment needs to be determined and the potential for therapeutically targeting vascular co-option evaluated.Normalisation of brain tumour vessels might be expected to help to restore the blood–brain barrier (BBB), which could be considered as an adverse effect on therapy, as this reduces the efficiency by which subsequent chemotherapy agents pass through the endothelium.Most FDA-approved anti-angiogenic drugs are primarily given when standard first-line cancer treatment has failed or when a patient is diagnosed with an advanced stage of the disease see Fig. . We also135Clinical trials involving breast cancer patients who received a single dose of bevacizumab followed by chemotherapy showed that the microvessel density prior to bevacizumab treatment could be used as a marker to predict treatment outcome—only those women whose tumours were highly vascularised before the treatment responded to bevacizumab-induced vessel normalisation.117 This association underscores the potential to use gene expression signatures associated with pericyte function to predict not merely vessel normalisation but perhaps also the efficacy of an associated immune response. As such it exemplifies the intimate relationship between vascular function and the immunological state of cancers.The genetic composition of tumour cells can determine whether or not a patient will respond to treatment by undergoing tumour vessel normalisation. So-called ‘Good Prognosis Angiogenesis Genes’ (GPAGs), discovered by screening breast cancer gene databases, are predominantly associated with cell–cell adhesion and smooth-muscle cell proliferation, and such gene expression programs are common in pericytes and pericyte recruitment in vessel normalisation GPAGs are also associated with immune response pathways, especially T-cell receptor signalling.136 In animal models, however, good results were obtained monitoring vessel normalisation using, for example, imaging biomarkers with fMRI or by photoacoustic imaging. Both methods were able to denote the normalisation window in mice and rats after low-dose anti-angiogenic treatment.137 The practical advantages of photoacoustic imaging over approaches that demand large and expensive instruments renders this technique a likely choice for monitoring the (tumour) vasculature in humans and in the clinic in the near future.138In humans, non-invasive monitoring down to the resolution of microvessels still remains a challenge. MRI, computed tomography (CT) and positron emission tomography (PET) scans are imaging methods that are routinely used in the clinic to measure the size and location of tumours and, although large vessel such as arteries and veins can be seen using a contrast agent (magnetic resonance angiogram or ‘MRA’), the spatial resolution is too low for microvessels, and the movement of fluids, essentially blood, cannot be detected. The clinical use of fMRI, which allows the measurement of blood oxygenation relative to the paramagnetic properties of deoxyhaemoglobin also lags behind its use in research.2) in murine tumours using electron paramagnetic resonance (EPR) and fMRI imaging identified a normalisation window 2–4 days after anti-angiogenic treatment with sunitinib. The perfusion of the tissue was improved and reduced the occurrence of acute hypoxia. The oxygen levels in the tissues were stabilised to normal and an oxidative shift of the tumour redox status in the metabolic pyruvate/lactate flux measured by an exogenous nitroxide probe and 13C-NMR was revealed.139 In principle, it would be possible to measure metabolic changes using PET—for example, fluorodeoxyglucose (FDG)-PET could be used to measure glucose uptake in endothelial cells. A reduced glucose uptake can lead to vessel normalisation and hence be a valuable indicator of this process.140 At the same time, a widespread goal is to step away from costly and time-consuming methods towards strategies that are more amenable to routine clinical practice.A further approach to determine the beginning and end of the vessel normalisation process involves non-invasive imaging of the tumour redox state and energy metabolism. Monitoring the partial pressure of oxygen , which, when activated, regulates blood pressure and encourages the formation of new blood vessels.144So much evidence now exists for the benefit of a normalised tumour vascular network as part of any cancer treatment that it offers an extraordinary opportunity to improve treatment outcomes. Immunotherapy, for instance, has progressed rapidly in the past five years, but not all types of tumour respond to this approach; it is now therefore vital to complement this treatment, and others, with well controlled adjustments to the vasculature. Finding the opportune moment to normalise the vasculature remains a challenge, and the discovery of a reliable and universally measurable indicator has yet to be made. However, some approaches to assess the problem are emerging. Measuring hypoxia-driven biomarkers, such as apelin, in a blood test might be more feasible than measuring the expression levels of a protein like CD109 in dormant endothelial cells or assessing blood oxygenation by fMRI each day over weeks.An additional challenge lies in how best to create such a ‘window of opportunity’ therapeutically and to keep it open for as long as necessary. Systemic and oral anti-angiogenic treatments can come with severe side effects. The range of clinically approved anti-angiogenic medication is limited, and most anti-angiogenic drugs will be given after conventional treatments have failed or if a patient is diagnosed with advanced stages of cancer see Fig. . Both fa81 Physical activity during or after cancer treatment has long been associated with overall well-being and improved quality of live. Now biological evidence can be added that increased blood flow will aid any following treatment and should be explored further. Nearly 2000 clinical trials are currently actively studying the behavioural impact of exercise on cancer patients, and valuable data can be obtained from these studies .Whereas the normalisation effect achieved by medication or radiotherapy can be short-lived and expensive, physical activity is cost-effective and discretionary, virtually free of side effects and applicable over extended periods of time. The benefit of creating a vessel normalisation window simply through moderate aerobic activity would be undeniable. The rate of activity that yielded promising results in exercising mice in a laboratory setting would translate to a daily brisk walk in humans, which is achievable even under life-changing circumstances.145 with evidence indicating that in-depth analyses of the vascular architecture and the dynamics in vascular function will provide the most robust indications regarding tumour adaptation. The classification of evolutionary stages in cancer would allow, for instance, a range of approved end-stage cancer therapies to be administered to patients at earlier stages of cancer without raising the risk for side effects.In conclusion, cancer researchers are increasingly appreciating that only a complete understanding of tumour plasticity and the evolutionary biology supporting tumorigenesis will achieve durable treatment responses,


Cutaneous burn injury is associated with epidermal loss in the zone of coagulation zone and delayed tissue loss in the zone of stasis. Thus, thermal stress can trigger both necrosis and regulated cell death (RCD) or apoptosis. Experimental in vitro and in vivo work has clearly demonstrated apoptotic events of thermally injured keratinocytes that are accompanied by morphological and biochemical markers of regulated cell death. However, in vivo data for the different pathways of regulated cell death are sparse. In vitro experiments with heat-stressed human keratinocytes have demonstrated death receptor involvement (extrinsic apoptosis), calcium influx, and disruption of mitochondrial membrane potential (intrinsic apoptosis) in regulated cell death. In addition, caspase-independent pathways have been suggested in regulated cell death. Keratinocyte heat stress leads to reduced proliferation, possibly as a result of reduced keratinocyte adhesion (anoikis) or oncogene involvement. Understanding the underlying mechanisms of RCD and the skin’s responses to thermal stress may lead to improved strategies for treating cutaneous burn trauma. Cutaneous burn injury is associated with epidermal loss that necessitates skin grafting in deep partial and full-thickness burns. Studies by Moritz and Henriques have analysed thermal stress to the skin and have demonstrated that the death of cells is time and temperature dependent . In 1953Cell death can be identified through biochemical or histopathological changes. Three different forms of histopathological alterations have to be distinguished: (1) type I cell death or apoptosis exhibiting cytoplasmic shrinkage, chromatin condensation (pyknosis), nuclear fragmentation (karyorrhexis), and plasma membrane blebbing; (2) type II cell death or autophagy manifesting with extensive cytoplasmic vacuolisation and phagocytic uptake and consequent lysosomal degradation; 3) type III cell death or necrosis with no distinctive features of type I or II cell death  type III.Further biochemical and functional research has deciphered the molecular basis of RCD, which has resulted in a more differentiated nomenclature . In addiMolecular features of apoptosis such as activation of endonucleases and degradation of DNA were also identified during terminal differentiation of keratinocytes in the epidermis . While tSpecifically, this review will focus on the impact of in vitro and in vivo thermal stress on keratinocyte death at the functional, molecular, and histopathological levels. Possible therapeutic interventions which may improve burn wound healing will be discussed. Understanding the underlying mechanisms and responses of the skin to thermal stress may lead to improved strategies for treating burn trauma.Physical contact with extremely high temperatures instantaneously results in accidental cell death with physical disassembly of the plasma membrane . NecrotiA variety of in vitro studies have evaluated the impact of heat on keratinocytes. The experiments involved HaCaT cells as well as primary keratinocytes. However, HaCaT cells resemble a spontaneously immortalised human keratinocyte cell line calling into question regular apoptotic pathways . It is w2+ depolarises the inner membrane of mitochondria, with a subsequent release of cytochrome c, formation of the apoptosome (with PARP-1), and cleavage of procaspase-9 to caspase 9 [Mitochondria play a central role in intrinsic apoptosis  3]. Mit. Mit3]. aspase 9 ,18. Heataspase 9 ,20. Openaspase 9 , which maspase 9 . In contaspase 9  reportedaspase 9 ,25. Cellaspase 9 . While taspase 9 .2+ release and apoptosis (see below)  h,27 h7,27in vitro , either in vitro  excludedin vitro  demonstrReddy et al.  reportedAnoikis is a specific variant of apoptosis initiated by the loss of integrin-dependent anchorage ,59. It iThe ultimate goal of understanding the mechanisms of cell death is to prevent tissue injury in pathophysiologic processes resulting from events such as thermal injury to the skin. A common question, therefore, is which form of cell death—apoptosis, necrosis, or a mixture—is occurring in this disease process. Currently, there are only a few immediate (on the scene) therapeutic interventions that are administered clinically to reduce heat-induced cell damage. Interestingly, these include cooling as well as warming . In vitrUnderstanding the mechanisms of cell death in cutaneous burn injury is a prerequisite for preserving keratinocytes of the epidermis and subsequently preventing sequelae, such as skin grafting or scarring. It is now well established that different modes of cell death occur after thermal injury to keratinocytes in vitro. Evidence from various groups indicates an intrinsic pathway involving mitochondria with downstream caspase-dependent apoptosis. However, there is still sparse information on whether these mechanisms of regulated cell death are of clinically significant relevance. Descriptive histological studies have proposed both caspase-dependent and caspase-independent pathways. Further in vivo proof-of-concept studies on blocking different pathways are warranted to decipher the precise pathophysiological relevance with functional and morphologic endpoints. In addition, further analyses of isolated keratinocytes are needed to delineate the signaling in order to determine the point of no return and timing of postburn interventions.


There has been a recent surge in interest in the role of inhaled corticosteroids (ICS) in the treatment of COPD, especially regarding patientswith high eosinophil counts. Evidence has shown that despite the increase in localised adverse effects and a small increase in non-fatalpneumonia events with ICS use, ICS still have an important role to play in reducing exacerbation rates and addressing the inflammationthat is at the heart of the pathogenesis of COPD. Current international guidelines recommend the use of ICS only in patients with severedisease. This review examines the potential role of ICS in all COPD patients. There aretwo primary reasons given against ICS use in other categories ofCOPD. Firstly, the GOLD strategy cites in vitro evidence that theinflammation present in COPD is inherently corticosteroid resistant.The second concern raised is the highly topical increased risk of nonfatal pneumonia in patients with severe COPD who use ICS.The use of inhaled corticosteroids (ICS) in chronic obstructivepulmonary disease (COPD) remains a topic of contention amongdoctors and data on the subject are often contradictory.Here, we review the evidence for the abovementioned assertions byexamining data on the efficacy of ICS in COPD, the pharmacologicalactions of ICS with relation to the pathogenesis of COPD, as wellas examining the strength of the evidence for an increased risk ofpneumonia in this population. We conclude with recommendationson the use of ICS.6] These effects are greaterin those who experience such events more frequently.8] There isgood evidence to show that long-term use of ICS reduces the rateof exacerbations in patients with both moderate and severe airflowlimitation.10]In patients with COPD, exacerbations are associated with anincreased risk of mortality, poorer quality of life, and acceleratedlong-term decline in lung function.[9] In this same meta-analysis it was noted that some studies also showed a reductionin rescue bronchodilator use.[9]ICS use has also been shown to affect patients’ quality of life (QOL)and symptoms. In a meta-analysis of ICS use for stable COPD, therate of decline in QOL as measured by the St George’s RespiratoryQuestionnaire (SGRQ) was reduced, and there was a small, butstatistically significant, improvement in patients’ QOL.et al.[11] suggested a slower rate of decline in the forced expiratoryvolume in one second (FEV1) in patients who received ICS therapy;however, long-term use of ICS has not consistently been shown toreduce the rate of decline in FEV1, or to have any significant effecton mortality in COPD patients.[9] These observations were mostrecently corroborated in the Study to Understand Mortality andMorbidITy in COPD (SUMMIT).[12]The Withdrawal of Inhaled Steroids during OptimizedBronchodilator Management (WISDOM) study by Magnusson1value <60% of predicted and a history of exacerbations, theSUMMIT sub-study suggests that ICS may also have a role in otherpatient groups, as there were benefits in those with an FEV1>60% ofpredicted and in patients with no exacerbation history.[10]While ICS are currently recommended for patients with anFEV[13] Long-acting muscarinic-antagonist(LAMA) and LABA therapy were independently ranked second andthird, and ICS alone was ranked fourth at 6 months. Martinez et al.[10]recently demonstrated that the combination of ICS/LABA reducedexacerbation rates to a greater degree than either component alone.It has been proposed that the mechanism of this synergistic effect isthe LABA enhancing glucocorticoid receptor nuclear translocationand efficacy. This was demonstrated in a study of induced-sputummacrophages: the combination of salmeterol and 100 µg fluticasonepropionate (FP) significantly increased nuclear glucocorticoid receptor levels equivalent to that of 500 µg FP, enhanced ICS-inducedmitogen-activated protein kinase phosphatase-1 (MAPK1) mRNAcopies and doubled glucocorticoid response element-luciferasereporter gene activity.[14] There is also evidence that the budesonide/formoterol combination enhanced the expression of pro-surfactantprotein-B in the lungs of COPD patients – a population in whichsurfactant expression is decreased and which has also been associatedwith poor health outcomes.[15]A meta-analysis of treatment options for patients with severe COPDwho remained uncontrolled on short-acting muscarinic-antagonists(SAMA) and short-acting beta-agonists (SABA) alone, found thata combination of an ICS and long-acting beta-agonist (LABA) wasthe highest-ranked intervention for improving QOL compared withplacebo at 6 and 12 months.Recently, data have emerged regarding the so-called ‘triple therapy’,which includes a combination of ICS/LAMA/LABA treatment.[16]Clinical trials have previously tested the effectiveness of tripletherapy delivered by two separate devices, compared to LAMAmonotherapy, LAMA/LABA combination therapy using separateinhalers, and combined ICS/LABA treatment. These studies showeda short-term superiority of triple therapy in terms of lung functionand patient-reported outcomes when compared with LAMAmonotherapy or ICS/LABA treatment.1in the group in which ICS was withdrawn,as well as a worsening of dyspnoea scores and health status outcomes.[11]It is important to note that this was a non-inferiority study and thusequivalence or superiority cannot be presumed.One study that compared triple therapy with LAMA/LABA combination therapy (the latter grouphaving had the ICS (FP) sequentially decreased and then completelywithdrawn from the initial triple regimen) noted no significantdifference in the exacerbation rate. However, they did observe asignificant decrease in FEV1, reduction in exacerbation rate, as well as health-related QOLscores. 17] In TRILOGY, there was a 23% reduction in exacerbationswith extra-fine beclomethasone dipropionate, formoterol furoateand glycopyrronium bromide (BDP/FF/GB) compared with BDP/FF.[16] In FULFIL , the addition of umeclidinium toFF/VI resulted in a net FEV1gain of 179 mL compared with BDP/FF at 1 year, with a higher percentage of subjects who were SGRQresponders in the former and a mean SGRQ change of –4.6 unitscompared with -1.9 U with BDP/FF.[17] Such a clinically significantdifference in the SGRQ  has seldom been documented in COPDtrials.Two large randomised trials have compared the ICS/LAMA/LABA combination in a single inhaler device with ICS/LABA, withsimilar results: in patients with severe COPD, triple therapy wasfound to be superior to ICS/LABA combination in improvements inFEV[18]Currently, there are no good-quality prospective data comparingtriple therapy with the LABA/LAMA combination.[19]A recent meta-analysis on the effects of withdrawal of ICS showedthat, while ICS withdrawal did not significantly increase the overallrate of COPD exacerbations, a clinically important increased riskof severe exacerbation was detected. ICS withdrawal significantlyimpaired both lung function and QOL. The time to the firstexacerbation was also significantly shorter in the patients whodiscontinued ICS.[20] The dominant inflammatory cellsare neutrophils; however, increased numbers of macrophagesand CD8+ T lymphocytes are also present, all of which interactto produce chemokines, cytokines, proteases and reactive oxygenspecies that cause tissue damage and stimulate further inflammation.22] The presence of inflammatory biomarkers in the sputumhas been associated with disease progression and an increased riskof exacerbations,[23] while suppression of airway inflammation hasbeen shown to improve lung function[24] and reduce exacerbationrates by up to 30%.[25]It is well known that inflammation of the airways is present evenin the early stages of COPD.[26]Corticosteroids suppress the multiple inflammatory genes thatare activated in chronic inflammatory diseases, such as COPD. Thisis achieved mainly by reversing histone acetylation of activatedinflammatory genes through binding of liganded glucocorticoidreceptors to coactivators, and recruitment of histone deacetylase-2(HDAC2) to the activated transcription complex.[27] However, it has been demonstrated that the actionof budesonide in suppressing airway inflammation is independentof the HDAC2 pathway.[28] Other postulated mechanisms of ICSresistance in COPD include activation of mitogen-activated protein(MAP) kinase pathways by certain cytokines, excessive activationof the transcription factor activator protein 1, raised macrophagemigration inhibitory factor, and increased P-glycoprotein-mediateddrug efflux.[29] However, a meta-analysis of studies examininginflammatory biomarkers in sputum, bronchoalveolar lavage fluidand biopsy specimens concluded that ICS were effective in reducingCD4+ and CD8+ T cell counts, as well as neutrophil and lymphocytecounts. It was noted that macrophage counts were increased in thepresence of ICS. The authors hypothesised that these importantimmunomodulatory effects could be the reason for the efficacy of ICSin reducing exacerbations, as well as the mechanism underlying theapparent increase in pneumonia.[29] A subsequent study concludedthat even in the presence of smoking, long-term ICS treatment maylead to anti-inflammatory effects in the lung as ICS reduced bronchialmast cells, CD3+, CD4+ and CD8+ cells, as well as sputum neutrophilsand lymphocytes.[30] In addition, a recent report has demonstrated thatICS discontinuation in patients on long-term ICS with moderate-to-severe COPD resulted in increased airway inflammation, as reflectedby increased numbers of bronchial CD3+, CD4+, and CD8+ T cells andmast cells, as well as increased sputum total cell count, macrophages,neutrophils and lymphocytes.[31]It has been suggested that the inflammation specific to COPD isresistant to corticosteroid effects, possibly through reduced HDAC2expression.[32] A COPD phenotypethat is more likely to respond to ICS has not yet been identified, asresponse is not predicted by oral steroid response, bronchodilatorreversibility or bronchial hyper-responsiveness.[32] However, thereis evidence that long-term benefits of ICS on lung function declinein patients with moderate-to-severe COPD are most pronounced in patients with fewer pack years smoking history, less severe emphysema(limited hyperinflation and preserved diffusion) and lower sputuminflammatory cell counts.[33]Another study identified an ICS-insensitive macrophage phenotypein COPD. These macrophages showed significantly lower expressionof all receptors, and were associated with higher levels of release ofactive matrix metalloproteinase 9 compared with macrophages ofnon-smokers and smokers without COPD.post hoc analysis of two large multinational studies comparingtreatment with ICS/LABA (fluticasone fuorate/vilanterol[VI]) toVI monotherapy, found that patients with a blood eosinophil count≥2.4%, responded better to the combination, with a generally linearrelationship of further exacerbation reduction with higher eosinophilcounts. The inference from their analysis was that, in general, loweosinophil counts coupled with high levels of smoking could predict apoorer response to ICS, with no significant reduction in exacerbationrates.[34] The linear association of blood eosinophils with exacerbation reduction by ICS was also noted in a further analysis of the WISDOMstudy using tiotropium, salmeterol and FP.[35] In a post hoc analysisof the INSPIRE (Investigating New Standards for Prophylaxis inReduction of Exacerbations) study using an eosinophil cut-off of 2%,FP/salmeterol was associated with a 25% relative risk reduction ofexacerbations compared with tiotropium alone.[36] This, and otherevidence regarding the anti-inflammatory effects of ICS in COPD, iscaptured in Table 145].A 46] Additionally,ICS increase the risk of non-fatal serious adverse pneumonia events, without conferring any differencein overall mortality rate.[47] This effectwas first unexpectedly identified in thelarge prospective TORCH  study,[48] andhas subsequently been shown in numerouslarge randomised trials (Table 2)63] andsmaller studies. Recent studies have reporteda stronger association with pneumonia athigher doses of ICS, suggesting a dose-response relationship, and age older than65 has been identified as an additionalfactor which increases risk.[46] However,it is worth noting that the incidence ofpneumonia in all the above studies is low. The latteris a reminder that COPD itself predisposespatients to pneumonia through an alteredmicrobiome and the toxic effects ofcigarette smoking, as well as the fact thatthe disease occurs in older individuals whomay have used oral corticosteroids, whichleads to further suppression of the immuneresponse.There is no doubt that the use of ICS is associated with an increasedrisk of localised adverse effects: oropharyngeal candidiasis, dysphoniaand hoarseness, as well as an increased risk of cataracts.65]The overall quality of the studies dedicatedto examining the treatment of COPD ishigh. There are a number of well-conductedrandomised controlled trials with largepatient numbers available for analysis.However, there have been a few criticismsof the studies from which the associationbetween ICS use and pneumonia is drawn.The prevailing hypothesis for themechanism of the propensity to pneumoniawith ICS is local airway immunosuppressionand a diminished innate immuneresponse to pathogens. Paradoxically, thisdiminished inflammatory effect is alsohypothesised to be the mechanism forthe lack of severity of these pneumoniaevents and thus the low fatality rate.[66]Dransfield et al.[52] reported a twofoldincrease in the pneumonia events inthe combination ICS/LABA (FF/VI)arm compared with the LABA (VI)monotherapy arm in patients with atleast one exacerbation in the previousyear and severe airflow obstruction. Theyrecruited >800 patients in each arm of the study, which in another context wouldbe considered a large study. However,the recent SUMMIT trial included >4000 patients in each arm (FF/VI v. themonotherapy components and placebo),with only moderate airflow obstruction, andreported that the difference in pneumoniaevents between the combination and theplacebo arm was not statistically significant.[10] These two studies highlight thedifficulties with interpretation of theexisting body of evidence: two large studiesreporting on the same outcome, about thesame drugs, but with different patientpopulations, vastly different numbers andopposing conclusions.The first criticism is a methodologicalone regarding differences in trial design,patient population and the type ofinterventions, all of which combine toincrease differences in the reported rateof pneumonia events between studies.[66]The second criticism is a ls omethodological in nature, and refers tothe case definition (or lack thereof) used inthe reporting of pneumonia events in thesetrials. The analysis of pneumonia eventswith ICS therapy in COPD is complicatedby the overlap in clinical features of COPDexacerbations and pneumonia, and by thefact that pneumonia remains a relativelyuncommon event when compared withacute exacerbations. While the majorityof these trials were randomised controlledtrials and had the highest quality evidence,they did not adhere to any formaldefinitions of pneumonia events. Rather,they relied on either the investigator’sretrospective assessment of a reportedadverse respiratory event or databasereporting systems. Chest radiographs werealso not routinely performed or assessed atthe time of the reported events.The recent suggestion that LAMA/LABA should be the baseline treatmentof all patients from GOLD B-D raises thefollowing two concerns:if inflammationis at the core of the pathogenesis of COPD,then this important component is notbeing addressed with bronchodilatorsalone; anddoes combination therapywith LAMA/LABA represent the ceilingeffect, that is, can no further therapeuticgain be achieved?[34] The search for amore reliable biomarker of the phenotype of ICS responsivenessis still underway.The available evidence shows that the useof ICS in patients with COPD does conferadditional clinically significant beneficialeffects, in particular the reduction ofexacerbations. While the inflammationthat occurs in COPD may be partially corticosteroid resistant, there is good evidence that the use of ICSreduces airway inflammation in a meaningful way. Furthermore,the data suggesting that certain COPD phenotypes will derivebenefit from ICS, particularly patients with blood eosinophilia≥2% or 150 cells/µL, is accumulating rapidly.[67] that, in considering ICS use, the riskbenefit should be carefully weighed. In one report, the exacerbationreduction with ICS use was in the order of 190 events, compared withthe minor increase of ~30 pneumonia events.[67] These and other dataprompted the European Medicines Agency’s Pharmacovigilance RiskAssessment Committee (PRAC) report to state that, while increasedrisk of pneumonia remains a common side-effect for all inhaledcorticosteroids, the benefits of ICS continue to outweigh the risk.[68]An increase in the risk of non-fatal pneumonia events has beendocumented; however, important methodological differences shouldbe considered when interpreting these trials. It is worth noting thatCOPD patients in these studies have a baseline risk of pneumoniaof up to 5.6% and the increase in risk is <2% Finally, given the promising nature of the emerging literature ontriple therapy, there is the possibility this may become the treatmentof choice in GOLD D patients, and may have a role in other categoriesof severity as well.More research is needed to identify the true ICS-responsivephenotype, as well as to assess the effects of triple therapy in earlystage COPD on lung function decline, as well as exacerbation rates.Careful attention will need to be paid to the rates of pneumonia andother adverse events in these patient groups so that an accurate riskbenefit assessment can be made, on an individual patient basis.


Vascular occlusive diseases such myocardial infarction, peripheral artery disease of the lower extremities, or stroke still represent a substantial health burden worldwide. In recent times, they have come even more into focus as thromboembolic events associated with vascular occlusive diseases are known to belong to the severe complications observed in patients with SARS-CoV-2 infection . To undeInternational Journal of Molecular Sciences entitled “Arteriogenesis and Therapeutic Angiogenesis” follows up with recent advances that are specific to that field of research.This Special Issue of the One of the most important points is to identify vascular occlusive diseases already in their beginning or early progression, enabling clinicians to induce natural bypass growth—a process that is defined as arteriogenesis or that is referred to as therapeutic angiogenesis—in time ,3. SinceMohamed Sabra and colleagues describe the mechanisms of vascularization, i.e. vasculogenesis, angiogenesis, and arteriogenesis, and critically elucidate the usefulness of angiogenic therapies such as protein therapy, gene therapy, stem cell therapy, and extracellular vesical therapy. Moreover, they address the relevance of patient selection and delivery methods, and introduce bioinformatics and bioengineering as promising future tools for the treatment of patients with vascular occlusive diseases . The papThe article by Ashraf and Zen is more oriented towards basic science and deals with the function of the quiescent contractile and the proliferative synthetic phenotypes of a vascular smooth muscle cell (VSMC) as well as the molecular mechanisms and regulations of phenotype switching. Moreover, the article critically addresses the option to target phenotype switching in patients aiming to promote arteriogenesis .Zeen Aref and Paul Quax highlight the difficulty of investigating the mechanisms of angiogenesis in hind limb ischemia models that are associated with arteriogenesis. They introduce an in vivo Matrigel plug assay, which is superior to current hind limb models, as it allows the analysis of ischemia-induced angiogenesis without the influence of collateral artery growth that is occurring in parallel . BesidesInternational Journal of Molecular Sciences, two groups have independently shown that the absence of CIRP promotes angiogenesis in ischemic muscle tissue, and, interestingly enough, they have identified two independent mechanisms. Kübler et al. have demonstrated that the absence of extracellular CIRP results in a reduced number of M1-like polarized pro-inflammatory and an increased number of M2-like polarized regenerative macrophages associated with reduced tissue damage and an increased capillary to muscle fiber ratio [Cold-inducible RNA-binding protein (CIRP or CIRBP) is a stress inducible protein that contains RNA and protein binding domains and that has recently come into the focus of vascular research ,11,12. Ier ratio . Goossener ratio , which her ratio .Yoshitomi and colleagues present the role of the AP-1 transcription factor family Jun B in angiogenesis and highlight its function in tip cell formation and tissue-specific vascular maturation .Eng+/− mice in a myocardial infarction model, Bakker et al. were able to show that these mice display more M1-like polarized macrophages, whereas the number of M2-like polarized macrophages was reduced. These data somehow reflect data from the clinic, as patients with HHT1 also show an increased number of inflammatory macrophages. Astonishingly, the treatment of Eng+/− mice with a bone morphogenetic protein (BMP) receptor kinase inhibitor improved heart function and vascularization, suggesting that the BMP receptor kinase may present a promising therapeutic target for HHT1 patients in the future [Endoglin is a co-receptor of transforming growth factor-β1 (TGF-β1), and mutations of this transmembrane protein are known to cause the vascular disorder hereditary hemorrhagic telangiectasia type 1(HHT1). By investigating e future .Gatina and co-workers investigated the serviceability of recombinant multicistronic mutagenic contructs in terms of safety and efficacy to treat ischemic disease. By using 2A-peptide-based constructs encoding vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), they were able to demonstrate increased levels of the named recombinant proteins along with an increased number of capillary-like structures in genetically modified human umbilical vein endothelial cells (HUVECs) in vitro . Last noIn summary, we think that our new Special Issue on arteriogenesis and therapeutic angiogenesis is a rewarding collection of original and review articles in the field of vascular research that will serve as inspiration for future pioneering investigations looking into the treatment of vascular occlusive diseases.


In menopausal women, the periodontium can be affected directly or through neural mechanism by oestrogen (E2) deficiency. The majority of the biological effects of E2 are modulated via both oestrogen receptor-α (ERα) and oestrogen receptor- β (ERβ). There is evidence that hypoestrogenism has a substantial impact on the aetiology, manifestation and severity of periodontitis, via the regulation of the expression of osteoprogesterin and RANKL in human periodontal ligament cells through ERβ. However, the mechanistic understanding of oestrogen in periodontal status has been partially clarified. The aim of this paper was to synopsize the recent scientific evidence concerning the link between the menopause and periodontitis, through the investigation of physio-pathological impact of the oestrogen deficiency on osteogenic differentiation of PDLSCs and PDLSC, as well as the dynamic change of ERα and ERβ. (2) Methods: Search was conducted for significant studies by exploring electronic PubMed and EMBASE databases, and it was independently performed by two researchers. All studies on the impact of oestrogen level on alveolar bone resorption were searched from 2005 to July 2020. Data selection was in concordance with PRISMA guidelines. (3) Results: Eight studies met the criteria and were included in this systematic review. All studies reported that oestrogen deficiency impairs the osteogenic and osteoblastic differentiation of PDL cells and oestrogen affects the bone formation capacity of cells. Seven studies were conducted on animal samples, divided into two groups: the OVX animals and animals who received the sham operation. (4) Conclusions: There is a multitude of data available showing the influence of menopause on periodontal status. However, the evidence of this line to investigation needs more research and could help explain the physiological linkage between menopause state and periodontal disease. The incidence and prevalence of periodontal disorders are subject to several factors, including the hormonal fluctuation ,2,3. InsMoreover, the characteristics of oestrogens deficiency include the induction of anthropogenic substances activity, distinguished by estrogenic properties. Among them, have been found a group of exogenous substance identified as endocrine disruptors to leach endocrine-disrupting chemicals, that interfere with bone remodelling process and calcium metabolism, exerting deleterious and pleiotropic effects on reproductive and bone tissues .Investigations concerning the oral microcirculation in postmenopausal women have revealed that the diameter of the loops, tortuosity of vessels in labial mucosa and density of periodontal mucosa are altered and predispose to periodontal inflammation. The swelling of endothelial cells and periocytes of the venules, adherence of granulocytes and platelets to vessel walls, increased vascular permeability and vascular proliferation have been associated with variations in the oestrogen levels ,7,8. TheThe impact of oestrogen deficiency state of menopause has gained growing attention. In this setting, this review was aimed to discuss the dynamic correlation between physiological changes during the menopause and the consequences on the alveolar bone microenvironment.A systematic review was performed using the PRISMA statement recommendations  21]..21].Searches were conducted in PubMed and Embase (through Ovid) (2005–2020) to identify relevant studies which investigated the relationship between menopause and periodontal status, and this search was independently performed by two researchers. The electronic searches were supplemented by manual searching of reference lists and reviews identify additional primary studies. The complete search strategy for each database is available in Two researchers separately searched for articles using the following terms: (menopause) OR  AND (oestrogen) AND (oestrogen receptor) AND (oestrogen receptor-α) AND (oestrogen receptor-β) AND  OR (periodontitis) AND  OR  OR (osseous density) OR (bone density) AND (RANK Ligand) OR (RANKL) AND (RANK) OR (RANK receptor) AND (osteoprotegerin) AND ) AND ) AND  AND periodontal ligament cells (hPDLSC).Studies that met the following criteria were included in the review: human and animal studies which focused on the impact of oestrogen levels on osteogenic differentiation of Periodontal Ligament cells (PDLC) and Stem Cells (PDLCs) of animal and human cell lines, and studies exclusion criteria were as follows: (1) reviews, comments and letters; (2) abstracts or thesis publications; (3) duplicated publications. Two investigators performed the study selection, data extraction and quality independently, and disagreements were resolved by discussions. The extracted information included the study type, first author’s name, publication year, characteristics of the study, and outcomes information. The risk of bias assessment tool was used to assess the quality of randomised controlled trials, by two authors separately.In total, the citation screening for the review on menopause age and periodontitis produced 115 manuscripts from PubMed and EMBASE. At the initial screening carried out for developing the comprehensive bibliographic literature database, 115 articles were identified from the sources. After excluding duplicates, the selected publications were scrutinized separately by two reviewers. One hundred papers were excluded based on the title and abstract. Then, 8 articles were finalized, as highlighted in The risk of bias for all studies is shown in Total scores ranged from 1 to 4 (max = 4). A quality score of 4 was assigned to 2 studies ,27. Onlyα (TNF). Several preclinical and clinical studies concerning the effect of E2 on the bone mesenchymal stem cell (BMSC) have shown to enhance bone proliferation and osteogenic differentiation in various species, including mouse, rat, and human [The majority of studies observed the microarchitecture deterioration in the alveolar bone that characterizes the osteoporosis. The association between the post-OVX period and Bone Mass Density (BMD) changes has been evaluated in several studies. A previous meta-analysis described a more notable decrease in BMD in animals with longer post-OVX periods. This systematic review identified publication bias for the outcomes of ERβ. In recent years, and especially in the last decade, the phenomenon of the relationship between periodontitis and systemic health is attracting a lot of attention around the research. A multidimensional paradigm is based on the view that periodontal inflammation could activate physiological changes and responses and, conversely, how systemic disorders may affect the periodontal attachment apparatus. It has been amply recognized that the incidence of periodontal disease increases rapidly remarked in menopausal women, and that the severity of periodontal tissue deterioration is more evident. The course of menopause is dictated by the ovarian function decline, accompanied by a substantial reduction in circulating sex steroid hormones. The in vitro study conducted by Mariotti A.J. , expandend human ,9,10. Hond human . It has nd human , six ovand human  emphasiznd human ,25,29 innd human  examinednd human ,32,33. Dnd human , extendend human  group rend human  that desnd human  focused nd human . Finallynd human  indicateThe present systematic review indicated menopause patients are more susceptible to experimental investigations have provided proof of increased prevalence of periodontal alveolar bone defects in presence of oestrogen deficiency. However, the mechanism by which oestrogen deficiency causes bone loss remains largely unknown. The translational validity of animal models of human menopause it is dubious, because of dissimilarity in bone remodelling conditions. Moreover, rodents do not suffer natural menopause.