Pathological Vessel Formation Research Articles

Vascular Heterogeneity With a Special Focus on the Hepatic Microenvironment.

Utilizing single-cell sequencing, recent studies were able to analyze at a greater resolution the heterogeneity of the vasculature and its complex composition in different tissues. Differing subpopulations have been detected, distinguishable only by their transcriptome. Designed to provide further insight into the heterogeneity of the functional vascular tissue, endothelial cells have been the main target of those studies. This review aims to present a synopsis of the variability of the different vascular beds, their endothelial variety, and the supporting cells that allow the vessels to serve their various purposes. Firstly, we are going to chart vascular tissue heterogeneity on a cellular level, describing endothelial diversity as well as stromal microenvironmental variety and interaction in a physiological setting. Secondly, we will summarize the current knowledge of pathological vessel formation in the context of cancer. Conventional anti-tumor therapeutic targets as well as anti-angiogenetic therapy is frequently limited by poor response of the tumor tissue. Reasons for moderate response and resistance to treatment can be found through different drivers of angiogenesis, different mechanisms of blood supply, but also in poorly understood tissue diversity. Based on this, we are comparing how pathologies alter the normal structure of vascular tissues highlighting the involved mechanisms. Lastly, illustrating the concept above, we will focus on the hepatic microenvironment, an organ of frequent metastatic spreading (e.g., from colorectal, breast, and lung cancers). We will address how the hepatic vasculature usually develops and subsequently we will describe how common liver metastases vary in their vasculature and the way they supply themselves (e.g., angiogenesis versus vessel co-option).

Topical Application of Hyaluronic Acid-RGD Peptide-Coated Gelatin/Epigallocatechin-3 Gallate (EGCG) Nanoparticles Inhibits Corneal Neovascularization Via Inhibition of VEGF Production.

Neovascularization (NV) of the cornea disrupts vision which leads to blindness. Investigation of antiangiogenic, slow-release and biocompatible approaches for treating corneal NV is of great importance. We designed an eye drop formulation containing gelatin/epigallocatechin-3-gallate (EGCG) nanoparticles (NPs) for targeted therapy in corneal NV. Gelatin-EGCG self-assembled NPs with hyaluronic acid (HA) coating on its surface (named GEH) and hyaluronic acid conjugated with arginine-glycine-aspartic acid (RGD) (GEH-RGD) were synthesized. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the antiangiogenic effect of GEH-RGD NPs in vitro. Moreover, a mouse model of chemical corneal cauterization was employed to evaluate the antiangiogenic effects of GEH-RGD NPs in vivo. GEH-RGD NP treatment significantly reduced endothelial cell tube formation and inhibited metalloproteinase (MMP)-2 and MMP-9 activity in HUVECs in vitro. Topical application of GEH-RGD NPs (once daily for a week) significantly attenuated the formation of pathological vessels in the mouse cornea after chemical cauterization. Reduction in both vascular endothelial growth factor (VEGF) and MMP-9 protein in the GEH-RGD NP-treated cauterized corneas was observed. These results confirm the molecular mechanism of the antiangiogenic effect of GEH-RGD NPs in suppressing pathological corneal NV.

IL27Rα Deficiency Alters Endothelial Cell Function and Subverts Tumor Angiogenesis in Mammary Carcinoma.

IL-27 regulates inflammatory diseases by exerting a pleiotropic impact on immune cells. In cancer, IL-27 restricts tumor growth by acting on tumor cells directly, while its role in the tumor microenvironment is still controversially discussed. To explore IL-27 signaling in the tumor stroma, we used a mammary carcinoma syngraft approach in IL27Rα-deficient mice. Tumor growth in animals lacking IL27Rα was markedly reduced. We noticed a decrease in immune cell infiltrates, enhanced tumor cell death, and fibroblast accumulation. However, most striking changes pertain the tumor vasculature. Tumors in IL27Rα-deficient mice were unable to form functional vessels. Blocking IL-27-STAT1 signaling in endothelial cells in vitro provoked an overshooting migration/sprouting of endothelial cells. Apparently, the lack of the IL-27 receptor caused endothelial cell hyper-activation via STAT1 that limited vessel maturation. Our data reveal a so far unappreciated role of IL-27 in endothelial cells with importance in pathological vessel formation.

Interleukin-17: The Role for Pathological Angiogenesis in Ocular Neovascular Diseases.

Ocular neovascular diseases are featured by abnormal angiogenesis in the eye, and they seriously threaten the human visual health. These diseases include proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), and retinal vein occlusion (RVO). In fact, ocular neovascular diseases represent the leading causes of vision impairment and blindness worldwide. Ocular neovascularization, the process of pathological vessel formation in eye, underlies ocular neovascular diseases. Cytokines have important regulatory roles in neovascularization through immunological networks. Interleukin (IL)-17, the signature cytokine produced by T helper 17 (Th17) cells, has proven to be involved in ocular neovascularization. However, roles of IL-17 in ocular neovascular diseases still remain controversial. This review provides an overview of the functional roles of IL-17 in ocular neovascular diseases from basic research to clinical evidence by focusing on PDR, AMD, ROP, and RVO. The possible roles of IL-17 in neovascularization are achieved through a regulatory network of cytoskeleton remodeling, vascular endothelial growth factor (VEGF), VEGF-related cytokines, and complement components. Current applications as well as potential therapies targeting IL-17 with genome editing systems are also outlined and discussed. Targeting IL-17 might be a promising therapeutic strategy against ocular neovascular diseases.

Endomucin is necessary for pathologic retinal neovascularization in vivo

Many ocular pathologies, including wet age‐related macular degeneration (AMD) and the retinopathy of prematurity, are characterized by abnormal neovascularization. Endomucin‐1 (EMCN), is an endothelial cell‐specific, sialic‐rich glycoprotein. We have shown that EMCN is necessary for VEGF‐mediated proliferation, migration and tube formation by human retinal endothelial cells in vitro and for normal retinal vessel development in vivo. Motivated by these finding, we examined the role of EMCN in the formation of pathological vessels using murine oxygen‐induced retinopathy (OIR) and the laser‐induced choroidal neovascularization (CNV) models. Intravitreal delivery of siEMCN to P12 mice following exposure to hyperoxia led to a significant reduction in avascular area (14.2±2.6% vs 22.6±4.7%, P<0.05) and neovascularization (2.6± 0.3% vs 3.5% ± 0.5%, P<0.05) in mice with EMCN knockdown at P17 compared to siCtrl mice. In the laser‐induced model of CNV, comparison of fluorescein angiograms at day 14 indicated that the CNV lesions in siEMCN‐treated mice were smaller and significantly less leaky than in mice (32.2±1.4% vs. 40.6±4.1%, P<0.05). Our data indicate EMCN reduction/neutralization may be a potential therapy for treating pathologic ocular neovascularization.Support or Funding InformationThis work was supported by EY5318 and EY26539 (PD'A), a Knight's Templar fellowship (CPW) and P30EY003790.

Diagnostic Value of Serum Angiogenesis Markers in Ovarian Cancer Using Multiplex Immunoassay.

As cancer development involves pathological vessel formation, 16 angiogenesis markers were evaluated as potential ovarian cancer (OC) biomarkers. Blood samples collected from 172 patients were divided based on histopathological result: OC (n = 38), borderline ovarian tumours (n = 6), non-malignant ovarian tumours (n = 62), healthy controls (n = 50) and 16 patients were excluded. Sixteen angiogenesis markers were measured using BioPlex Pro Human Cancer Biomarker Panel 1 immunoassay. Additionally, concentrations of cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) were measured in patients with adnexal masses using electrochemiluminescence immunoassay. In the comparison between OC vs. non-OC, osteopontin achieved the highest area under the curve (AUC) of 0.79 (sensitivity 69%, specificity 78%). Multimarker models based on four to six markers (basic fibroblast growth factor—FGF-basic, follistatin, hepatocyte growth factor—HGF, osteopontin, platelet-derived growth factor AB/BB—PDGF-AB/BB, leptin) demonstrated higher discriminatory ability (AUC 0.80–0.81) than a single marker (AUC 0.79). When comparing OC with benign ovarian tumours, six markers had statistically different expression (osteopontin, leptin, follistatin, PDGF-AB/BB, HGF, FGF-basic). Osteopontin was the best single angiogenesis marker (AUC 0.825, sensitivity 72%, specificity 82%). A three-marker panel consisting of osteopontin, CA125 and HE4 better discriminated the groups (AUC 0.958) than HE4 or CA125 alone (AUC 0.941 and 0.932, respectively). Osteopontin should be further investigated as a potential biomarker in OC screening and differential diagnosis of ovarian tumours. Adding osteopontin to a panel of already used biomarkers (CA125 and HE4) significantly improves differential diagnosis between malignant and benign ovarian tumours.

Ephrin B2 and EphB4 selectively mark arterial and venous vessels in cerebral arteriovenous malformation

Ephrin type B receptor 4 (EphB4, Eph receptor) selectively binds ephrin B2 (Eph ligand). EphB4/ephrin B2 is involved in embryonic vessel development, vascular remodelling and pathological vessel formation in adults (including tumour angiogenesis). Binding of vascular endothelial growth factor (VEGF)-A to the endothelial-specific receptor VEGF receptor-2 is the main extracellular signal triggering angiogenic response. Little is known about the role of EphB4/ephrin B2 during angiogenesis and arteriovenous plasticity in cerebral arteriovenous malformation (cAVM). This study investigated EphB4 and ephrin B2 expression in cAVM. Haemorrhagic (H-AVM) and nonhaemorrhagic (NH-AVM) specimens of AVM nidus, obtained after microsurgical cAVM resection, and normal superficial temporal artery (STA) specimens, were analysed retrospectively. VEGF-A, EphB4 and ephrin B2 expression were studied by immunohistochemistry and immunoblotting. In cAVM (10 H-AVM; 10 NH-AVM), VEGF-A was immunocytochemically localized to endothelial cells; strong endothelial cell staining was found for EphB4 in veins and ephrin B2 in arteries. Normal STA (n = 10) did not express EphB4 or ephrin B2. EphB4 and ephrin B2 expression was greater in H-AVM than in NH-AVM. Endothelial cells are more active in H-AVM than NH-AVM. EphB4 and ephrin B2 play important roles in neovascularization and arteriovenous differentiation/plasticity. These data provide new insights into the aetiology of cAVM and lay a foundation for further study. The notch pathway induced by VEGF-A may be a key signalling pathway in this process.

Angiotensin II modulates VEGF-driven angiogenesis by opposing effects of type 1 and type 2 receptor stimulation in the microvascular endothelium

Vascular endothelial growth factor (VEGF) is a main stimulator of pathological vessel formation. Nevertheless, increasing evidence suggests that Angiotensin II (Ang II) can play an augmentory role in this process. We thus analyzed the contribution of the two Ang II receptor types, AT1R and AT2R, in a mouse model of VEGF-driven angiogenesis, i.e. oxygen-induced proliferative retinopathy. Application of the AT1R antagonist telmisartan but not the AT2R antagonist PD123,319 largely attenuated the pathological response. A direct effect of Ang II on endothelial cells (EC) was analyzed by assessing angiogenic responses in primary bovine retinal and immortalized rat microvascular EC. Selective stimulation of the AT1R by Ang II in the presence of PD123,319 revealed a pro-angiogenic activity which further increased VEGF-driven EC sprouting and migration. In contrast, selective stimulation of the AT2R by either CGP42112A or Ang II in the presence of telmisartan inhibited the VEGF-driven angiogenic response. Using specific inhibitors (pertussis toxin, RGS proteins, kinase inhibitors) we identified G12/13 and Gi dependent signaling pathways as the mediators of the AT1R-induced angiogenesis and the AT2R-induced inhibition, respectively. As AT1R and AT2R stimulation displays opposing effects on the activity of the monomeric GTPase RhoA and pro-angiogenic responses to Ang II and VEGF requires activation of Rho-dependent kinase (ROCK), we conclude that the opposing effects of the Ang II receptors on VEGF-driven angiogenesis converge on the regulation of activity of RhoA-ROCK-dependent EC migration.

Clinical Summaries

Clinical Summaries

Angiopoietin-1 and Myeloma-Induced Angiogenesis

Multiple myeloma (MM) is a plasma cell malignancy characterized by an increase of the bone marrow angiogenesis. Angiopoietin-1 (Ang-1) is a critical factor in the regulation of physiological and pathological vessel formation that acts by binding to a specific receptor Tie2 expressed on endothelial cells. Recent evidences indicate that human MM cells produce Ang-1 and up-regulate its receptor Tie2 in bone marrow endothelial cells. An overexpression of Ang-1 has been also found in MM cells as compared to normal plasma cells. The correlation between Ang-1 expression and BM angiogenesis, demonstrated in MM patients, and the inhibitory effect of Tie2 blocking on MM-induced vessel formation suggest that Ang-1 production by MM cells is critically involved in the angiogenic process in MM. In this review we focalize our attention on Ang-1/Tie2 system and its role in MM-induced angiogenesis.

Elevated serum vascular endothelial growth factor is associated with visceral fat accumulation in human obese subjects.

Adipose tissue expresses some bioactive molecules, which may be involved in the development of obesity-associated metabolic disorders and cardiovascular diseases. Vascular endothelial growth factor (VEGF) an important angiogenic factor is implicated in normal and pathological vessel formation. The aim of this study is to investigate clinically the association between blood serum VEGF concentrations and body fat accumulation as well as distribution. The study also aims to show the effect of serum VEGF protein on gene expression of transcriptional factor E26 transformation-specific-1 (Ets-1) and matrix metalloproteinase (MMP)-3. Serum VEGF concentrations were measured in 38 overweight or obese subjects. Fat distribution in the abdominal subcutaneous as well as visceral fat areas was assessed by computed tomography scans at umbilical level. Furthermore, the changes of serum VEGF concentrations following body weight reduction therapy were analyzed in eight subjects recruited from the original pool of subjects. Semi-purified circulating VEGF proteins were obtained by heparin-sepharose and its biological activities were shown to alter gene expressions in human aortic endothelial cells. Serum VEGF concentrations were positively correlated with BMI (r=0.433, p=0.007) and visceral fat area (r=0.488, p=0.002). Stepwise regression analysis showed the visceral fat area as the most important determinant factor for VEGF circulating levels. Following body weight reduction therapy, VEGF concentrations as well as visceral fat area were decreased. The serum semi-purified VEGF protein enhanced expressions of Ets-1 and MMP-3 in human aortic endothelial cells. Increased serum VEGF concentrations associated with visceral fat accumulation could influence vascular endothelial function.

Vascular development and disorders: Molecular analysis and pathogenic insights

Blood vessels are among the first organs to develop during embryogenesis and are essential for organogenesis and nutrition of the embryo. Although formation of blood vessels most actively occurs during embryonic development, tissue vascularization proceeds after birth in the retina, in the heart, and, cyclically, in the reproductive organs. In addition, abnormal vessel growth importantly contributes to the pathogenesis of several disorders with high morbidity and mortalitiy. Excessive vessel growth has been implicated in the pathogenesis of retinopathies, cancer and inflammation. In contrast, insufficient vessel growth may lead to tissue ischemia and failure. Although formation of new blood vessels has typically been associated with endothelial cells, the periendothelial mural pericytes (smaller vessels) or smooth muscle cells (larger vessels) are equally important for normal and pathological vessel formation. Indeed, a developing network of endothelial tubes that is not surrounded by mural cells is fragile and prone to rupture, remains susceptible to hypoxic regulation, fails to become remodeled, is unable to sustain proper circulation, and cannot adapt to changes in physiological demands of blood supply. Notably, vascular growth does not always involve formation of new capillaries, but may depend on remodeling of pre-existing arteriolized vessels, such as during collateral growth in the myocardium. The importance of periendothelial cells is further deduced from the pathological loss of smooth muscle cells in atherosclerotic vessels (leading to media necrosis), or from the pericyte drop out in diabetic retinal vessels, which renders them prone to aneurysmal rupture. Conversely, accumulation of smooth muscle cells contributes to atherosclerotic plaque growth and arterial stenosis. Recent studies have highlighted the importance of endothelial periendothelial cell crosstalk during normal and pathological blood vessel formation. Accordingly, integrated research into the molecular mechanisms that regulate the development and function of both endothelial and pericyte/smooth muscle cells has become a major focus in vascular biology. A number of candidate molecules (growth factors, matrix component, adhesion molecules, and proteinases) have been identified that stimulate or inhibit these processes. Recently, remarkable progress in their molecular analysis has been achieved through targeted manipulation of the mouse genome. Their role will be discussed in this review.

Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system.

VEGF has been proposed to participate in normal and pathological vessel formation. Surprisingly, lack of only a single VEGF allele resulted in embryonic lethality due to abnormal formation of intra- and extra-embryonic vessels. Homozygous VEGF-deficient embryos, generated by tetraploid aggregation, revealed an even more severe defect in vessel formation. These results (1) suggest a tight regulation of early vessel development by VEGF and, indirectly, the presence of other VEGF-like molecules; (2) reveal an unprecedented lethal phenotype associated with heterozygous deficiency of an autosomal gene, and (3) demonstrate that tetraploid aggregation was a valid and the only method to study the phenotype of the homozyogous VEGF-deficient embryos. The dominant and strict dose-dependent role of VEGF in vivo renders this molecule a desirable therapeutic target for promoting or preventing angiogenesis. Tissue factor (TF) is the principal cellular initiator of coagulation and its deregulated expression has been related to thrombogenesis in sepsis, cancer, and inflammation. However, TF appears to be also involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis. Surprisingly, TF deficiency resulted in embryonic lethality due to abnormal extra-embryonic vessel development and defective vitelloembryonic circulation. The abnormal yolk sac vasculature is reminiscent of that observed in embryos lacking VEGF, possibly suggesting that both gene functions are interconnected. These targeting studies extend the recently documented role of TF in tumor-associated angiogenesis and warrant further study of its role in angiogenesis during other pathological disorders. The plasminogen system, via its triggers, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis. Studies in mice with targeted gene inactivation of t-PA, u-PA, PAI-1, the urokinase receptor (u-PAR), and plasminogen (Plg) revealed (1) that deficiency of t-PA or u-PA increase the susceptibility to thrombosis associated with inflammation and that combined deficiency of t-PA:u-PA or deficiency of Plg induces severe spontaneous thrombosis; (2) that vascular injury-induced neointima formation is reduced in mice lacking u-PA-mediated plasmin proteolysis, unaltered in t-PA- or u-PAR-deficient mice and accelerated in PAI-1-deficient mice, but that it can be reverted by adenoviral PAI-1 gene transfer; and (3) that atherosclerosis in mice doubly deficient in apolipoprotein E (apoE) and PAI-1 is reduced after 10 weeks of cholesterol-rich diet. Thus, the plasminogen system significantly affects thrombosis, restenosis, and atherosclerosis.

Molecular weight and concentration of hyaluronan in vitreous humour from diabetic patients

Abstract Vitreous material was collected from 24 patients undergoing pars plana vitrectomy due to hemorrhage, proliferation or traction in the vitreous body or retinal detachment. Fifteen patients had a proliferative diabets retinopathy. The non‐diabetic group consisted of four patients with vascular disease: periphlebits (2), and branch vein occlusion (2) and five patients with a non‐vascular disease: retinal detachment (4) and macular pucker (1). Hyaluronan analyses of the samples, which constituted about 80% of the vitreous bodies, gave values from 3 to 2600 μg, corresponding to approximately 1–700 μg/ml in situ. Low values were recorded in patients operated on for cataract and especially in those who also had retinal detachment. Molecular weight distributions of hyaluronan were determined in 8/15 diabetics and in 5/9 non‐diabetics. Due to the low hyaluronan content it was not possible to perform molecular weight determination in more than one of the patients with retinal detachment. The hyaluronan was of high molecular weight in both diabetics and non‐diabetics; the weight‐average molecular weight (Mw) being 1.6–6.1 times 106 and 1.0–5.3 times 106, respectively. From this investigation there is no evidence that a change in the molecular properties of hyaluronan could be the cause of proliferative diabetic retinopathy.The vitreous humour contains a comparatively high concentration of hyaluronan (HYA) (100–400 μg/g). It is of high molecular weight and does not normally seem to be degraded extracellularly in the eye (Balazs & Denlinger, 1984).Proliferative diabetic retinopathy is characterized by pathological vessel formation. Recent investigations have shown that HYA can effect angiogenesis. High‐molecular weight HYA seems to inhibit blood vessel formation (Feinberg & Beebe 1983, Dvorak & al 1987, West & Kumar, 1989), while oligosaccharides produced by degradation of HYA induce an angiogenic response in the chick chorioallantoic membrane (West et al, 1985). West & Kumar (1989) have also shown in vitro that vascular endothelial cells endocytose HYA, probably mediated by a receptor. They have proposed the hypothesis that vasoproliferation in diabetic patients is brought about by a change in concentration and molecular size of HYA (West & Kumar, 1988). This led us to test the hypothesis that hyaluronan is degraded in the diabetic eye and that the degradation products could be at least partly responsible for vascularization of the vitreous body. However, we find no difference in the molecular weight of HYA in diabetic and non‐diabetic patients.

The angiographic investigation of duodenal tumours (author's transl)

The diagnosis and differential diagnosis of primary duodenal carcinomas by conventional radiological methods is difficult. Valuable information can be obtained by angiography, since primary duodenal carcinomas, unlike carcinomas of the papilla and pancreas, are characterised by the formation of pathological vessels arising from the duodenal branches of the gastro-duodenal artery. The integrity of the latter is an important differentiating feature. Exact localisation of the tumour is important in pre-operative assessment and diagnosis.