VEGFR-3 and Tie pathways in vascular network formation

The blood and lymphatic vascular systems are essential for life, but they may become harnessed for sinister purposes in pathological conditions. For example, tumors learn to grow a network of blood vessels (angiogenesis), securing a source of oxygen and nutrients for sustained growth. On the other hand, damage to the lymph nodes and the collecting lymphatic vessels may lead to lymphedema, a debilitating condition characterized by peripheral edema and susceptibility to infections. Promoting the growth of new lymphatic vessels (lymphangiogenesis) is an attractive approach to treat lymphedema patients. Angiopoietin-1 (Ang1), a ligand for the endothelial receptor tyrosine kinases Tie1 and Tie2. The Ang1/Tie2 pathway has previously been implicated in promoting endothelial stability and integrity of EC monolayers. The studies presented here elucidate a novel function for Ang1 as a lymphangiogenic factor. Ang1 is known to decrease the permeability of blood vessels, and could thus act as a more global antagonist of plasma leakage and tissue edema by promoting growth of lymphatic vessels and thereby facilitating removal of excess fluid and other plasma components from the interstitium. These findings reinforce the idea that Ang1 may have therapeutic value in conditions of tissue edema. VEGFR-3 is present on all endothelia during development, but in the adult its expression becomes restricted to the lymphatic endothelium. VEGF-C and VEGF-D are ligands for VEGFR-3, and potently promote lymphangiogenesis in adult tissues, with direct and remarkably specific effects on the lymphatic endothelium in adult tissues. The data presented here show that VEGF-C and VEGF-D therapy can restore collecting lymphatic vessels in a novel orthotopic model of breast cancer-related lymphedema. Furthermore, the study introduces a novel approach to improve VEGF-C/VEGF-D therapy by using engineered heparin-binding forms of VEGF-C, which induced the rapid formation of organized lymphatic vessels. Importantly, VEGF-C therapy also greatly improved the survival and integration of lymph node transplants. The combination of lymph node transplantation and VEGF-C therapy provides a basis for future therapy of lymphedema. In adults, VEGFR-3 expression is restricted to the lymphatic endothelium and the fenestrated endothelia of certain endocrine organs. These results show that VEGFR-3 is induced at the onset of angiogenesis in the tip cells that lead the formation of new vessel sprouts, providing a tumor-specific vascular target. VEGFR-3 acts downstream of VEGF/VEGFR-2 signals, but, once induced, can sustain angiogenesis when VEGFR-2 signaling is inhibited. The data presented here implicate VEGFR-3 as a novel regulator of sprouting angiogenesis along with its role in regulating lymphatic vessel growth. Targeting VEGFR-3 may provide added efficacy to currently available anti-angiogenic therapeutics, which typically target the VEGF/VEGFR-2 pathway.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

Polymorphisms in the vascular endothelial growth factor gene and the risk of familial endometriosis

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein suspected to be involved in the pathogenesis of endometriosis by establishing a new blood supply to the human exfoliated endometrium. Several transcription factor-binding sites are found in the VEGF 5'-untranslated region and variation within the region increases the transcriptional activity. Six previous studies which tested between one and three single nucleotide polymorphisms (SNPs) in samples comprising 105-215 cases and 100-219 controls have produced conflicting evidence for association between the SNPs in the VEGF region and endometriosis. To further investigate the reported association between VEGF variants and endometriosis, we tested the four VEGF polymorphisms (-2578 A/C, rs699947; -460 T/C, rs833061; +405 G/C, rs2010963 and +936 C/T, rs3025039) in a large Australian sample of 958 familial endometriosis cases and 959 controls. We also conducted a literature-based review of all relevant association studies of these VEGF SNPs in endometriosis and performed a meta-analysis. There was no evidence for association between endometriosis and the VEGF polymorphisms genotyped in our study. Combined association results from a meta-analysis did not provide any evidence for either genotypic or allelic association with endometriosis. Our detailed review and meta-analysis of the VEGF polymorphisms suggests that genotyping assay problems may underlie the previously reported associations between VEGF variants and endometriosis.

Exercise and vascular function in child obesity: A meta-analysis

CONTEXT: Conduit artery flow-mediated dilation (FMD) is a noninvasive index of preclinical atherosclerosis in humans. Exercise interventions can improve FMD in both healthy and clinical populations. OBJECTIVE: This systematic review and meta-analysis aimed to summarize the effect of exercise training on FMD in overweight and obese children and adolescents as well as investigate the role of cardiorespiratory fitness (peak oxygen consumption [Vo2peak]) on effects observed. DATA SOURCES: PubMed, Medline, Embase, and Cinahl databases were searched from the earliest available date to February 2015. STUDY SELECTION: Studies of children and/or adolescents who were overweight or obese were included. DATA EXTRACTION: Standardized data extraction forms were used for patient and intervention characteristics, control/comparator groups, and key outcomes. Procedural quality of the studies was assessed using a modified version of the Physiotherapy Evidence Base Database scale. RESULTS: A meta-analysis involving 219 participants compared the mean difference of pre- versus postintervention vascular function (FMD) and Vo2peak between an exercise training intervention and a control condition. There was a significantly greater improvement in FMD (mean difference 1.54%, P < .05) and Vo2peak (mean difference 3.64 mL/kg/min, P < .05) after exercise training compared with controls. LIMITATIONS: Given the diversity of exercise prescriptions, participant characteristics, and FMD measurement protocols, varying FMD effect size was noted between trials. CONCLUSIONS: Exercise training improves vascular function in overweight and obese children, as indicated by enhanced FMD. Further research is required to establish the optimum exercise program for maintenance of healthy vascular function in this at-risk pediatric population.

CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.