Angiogenesis

There is a need for simple, reproducible methodology for measurement of endothelial cell (EC) apoptosis and proliferation in formalin-fixed, paraffin-embedded (FFPE) human tissue obtained in clinical trials of potential anti-angiogenic agents. Therefore, we developed colorimetric, dual-label immunohistochemical techniques for use on FFPE tissue, based on the use of single-stranded (ss) DNA and Ki-67 as markers of EC apoptosis and proliferation, respectively. Digital image analysis was used to obtain the total tumour microvessel density (MVD), from which the EC apoptosis index (AI) and proliferation index (PI) were derived manually as the number of positive ECs per vessel. Immunohistochemical measurement of EC apoptosis and proliferation was validated on human colorectal cancer liver metastases from a randomized, placebo-controlled trial of the selective cyclooxygenase-2 inhibitor rofecoxib. Proliferating and apoptotic ECs clustered in discrete areas of the tumour vasculature. ECAI (median [inter-quartile range, IQR] 0.0018 [0.0003–0.0064]) and ECPI (median [IQR] 0.0043 [0.002–0.014]) values were low and highly variable in tumours from both placebo- and rofecoxib-treated patients. Our novel dual-label immunohistochemical methods will be generally applicable in FFPE human cancer tissue and should prove invaluable for measuring the anti-angiogenic activity of experimental therapies in clinical trials. The low absolute level of and variability in EC apoptosis and proliferation detectable in human colorectal cancer liver metastases indicates that similar intervention studies using these end-points will need to ensure adequate size in order to be able to detect anti-angiogenic activity by immunohistochemical methods.

Blood vessels demonstrate a multitude of complex signaling programs that work in concert to produce functional vasculature networks during development. A known, but less widely studied, area of endothelial cell regulation is vesicular trafficking, also termed sorting. After moving through the Golgi apparatus, proteins are shuttled to organelles, plugged into membranes, recycled, or degraded depending on the internal and extrinsic cues. A snapshot of these protein-sorting systems can be viewed as a trafficking signature that is not only unique to endothelial tissue, but critically important for blood vessel form and function. In this review, we will cover how vesicular trafficking impacts various aspects of angiogenesis, such as sprouting, lumen formation, vessel stabilization, and secretion, emphasizing the role of Rab GTPase family members and their various effectors.

The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach.

Vascular angiogenesis is regulated by a number of cytokines of which vascular endothelial growth factor (VEGF)-A/and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) play an indisputable role. Similarly lymphangiogenesis is regulated by VEGF-C and its receptor VEGFR3. Currently for treating vasculogenesis diseases such as proliferative retinopathies and cancer, a number of anti-VEGF-A therapies are approved for clinical use. Although clinical efficacies achieved are remarkable, they are found to be transitory in nature, followed by restoration of anti-VEGF therapy resistant angiogenesis. Recently the regulatory role of VEGF-C in initiating and potentiating neo-angiogenesis has been uncovered. Although the interactive nature of VEGF-A and C is known, the dynamics of their expression under knockdown conditions is yet to be established. Here in this study we have utilized siRNA to knockdown both VEGF-A and C either independently or in combination. Analysis of VEGF-A and C expression (only in cancer cell lines MCF7, A549 and H460 but not in the ocular cell line RPE19) has shown enhanced expression levels of VEGF-C with increase in knockdown of VEGF-A. However, VEGF-C knockdown has resulted in decreased expression levels of VEGF-A both in RPE19 and MCF7 cells in a dose dependent manner. In addition, VEGF-C knockdown also resulted in decreased expression of RhoA. Further, knockdown studies of RhoA even with supplementation of VEGF-C or A has resulted in decreased endothelial cell proliferation and stress fiber formation, indicating that VEGF-C does promote angiogenesis via RhoA mediated pathway.

Substantial weight loss through intense dietary regimens is thought to ameliorate endothelial dysfunction in obesity. It is less clear whether similar improvements can be achieved with modest dietary interventions. This study aimed to identify the parameters of endothelial cell status in obesity that are affected by mild calorie restriction. Human umbilical vein endothelial cells (EA.hy926 line) in culture were exposed pairwise to serum from 57 individuals with simple obesity (BMI > 30 kg/m2) collected before and after 8-week dietary intervention with energy deficit of 300–500 kcal/day. Analysis of endothelial transcriptome suggested that the intervention could impact on endothelial cell growth. Cell proliferation was measured with the MTT test and verified by [3H]-thymidine incorporation. The participants were categorized according to a change in proliferation over time. Significant decrease in endothelial cell proliferation correlated with the extent of weight loss in men, but not in women. This effect corresponded with changes in serum levels of leptin and adiponectin, but was not related to serum concentrations of several known angiogenic mediators (VEGF, MCP-1, TSP-1, MMP-9, angiopoietin-2). Direction and magnitude of changes in serum-induced endothelial cell proliferation identifies patients with the greatest weight loss in response to modest calorie restriction.

The Tie2 receptor and its known ligands, the angiopoietins, play a critical role in endothelial cell differentiation during the process of angiogenesis. Recent experimental observations indicate that the agonistic ligand, angiopoietin-1, can stimulate endothelial cell sprouting and act as a chemo-attractant in vitro and induce increased and enhanced angiogenesis both alone and in conjunction with vascular endothelial growth factor (VEGF) in vivo. Here, we present a monoclonal antibody (MAb), which binds to the extracellular portion of the Tie2 receptor and elicits similar agonist effects. Upon MAb binding to the native Tie2 receptor of cultured human umblical vein endothelial cells (HUVEC), there is a rapid increase in receptor autophosphorylation with a concomitant enhancement in the recruitment and association of the signalling intermediates Grb2 and SH-PTP2. The antibody further demonstrates functional activity in vascular tissues. In vitro, the antibody promotes the survival of cultured HUVEC and elicits a dose dependent outgrowth and branching of microvessels from cultured explants of rat aorta. When administered in vivo, the antibody enhances the vascularization of subcutaneous Matrigel implants in mice. Together these data suggest that the antibody is capable of acting as a surrogate ligand for Tie2 and further confirms the role of Tie2 in the differentiation of endothelial cells during angiogenesis.

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD is characterized by an excessive cerebral amyloid deposition leading to degeneration of neurons and eventually to dementia. It has been shown by epidemiological studies that cardiovascular drugs with an anti-angiogenic effect can influence the outcome of AD patients. Therefore, it has been speculated that in AD angiogenesis in the brain vasculature may play an important role. Here we report that in the brain of APP23 mice – a transgenic model of AD – after deposition of amyloid in blood vessels endothelial cell activation occurs in an age-dependent manner. Amyloid deposition is followed by the expression of β3-integrin, a specific marker molecule of activated endothelium. The β3-integrin expression is restricted to amyloid-positive vessels. Moreover, homogenates of the brains of APP23 mice induced the formation of new vessels in an in vivo angiogenesis assay. Vessel formation could be blocked by the VEGF antagonist SU 4312 as well as by statins, suggesting that these drugs may interfere with endothelial cell activation in AD. In conclusion our results indicate that amyloid deposition in the vasculature leads to endothelial cell apoptosis and endothelial cell activation, which can be modulated by anti-angiogenic drugs.

Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor with potent angiogenic activity and a stimulator of microvascular permeability. Because endometrial cyclic development is associated with vascular growth, we examined the expression of VEGF protein throughout the menstrual cycle and studied the regulation of VEGF mRNA by ovarian steroids in isolated human endometrial stromal cells. VEGF was localized immunohistochemically in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles, a localization which has not been described before. The strongest immunoreactivity for VEGF on endothelial cells was detected in the late proliferative and secretory phases. The localization of VEGF bound to the endothelium correlates with the presence of flt-1 and flk/KDR receptors on vascular structures, including capillary strands which have not yet formed a lumen, present during the mid-secretory period, which corresponds to a high estroprogestin influence and to implantation. Heparinase treatment of the sections decreases the staining intensity of VEGF bound to endothelial cells, suggesting that VEGF also binds to heparin-like molecules on the cell surface. These new results demonstrate a major role of VEGF on capillary formation and on hyperpermeability and edema during the menstrual cycle. Consistent with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E2) or E2 plus progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner; the VEGF mRNA response to E2 was rapid (3h) and persisted with continuous estradiol treatment up to 12 days. Three species, VEGF121, VEGF165 and VEGF189, were observed upon hormonal stimulation. The estradiol up-regulation of VEGF response did not require de novo protein synthesis as it was not blocked by cycloheximide. Also, the ability of the pure anti-estrogen ICI 182,780 to significantly block induction of VEGF mRNA by E2 suggests estrogen receptor-mediated transcriptional regulation. These results demonstrate that VEGF is an estrogen-responsive angiogenic factor that acts on vascular endothelium in a paracrine fashion, as previously suggested. This growth factor controls angiogenesis and hyperpermeability required for adequate receptivity to implantation of the cycling human endometrium. These findings also raise the possibility that estrogen effects on uterine edema, proliferation and tumoral transformation may involve local increases in tissue VEGF production.