Ruslan Hlushchuk

Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling

New blood vessels arise initially as blood islands in the process known as vasculogenesis or as new capillary segments produced through angiogenesis. Angiogenesis itself encompasses two broad processes, namely sprouting (SA) and intussusceptive (IA) angiogenesis. Primordial capillary plexuses expand through both SA and IA, but subsequent growth and remodeling are achieved through IA. The latter process proceeds through transluminal tissue pillar formation and subsequent vascular splitting, and the direction taken by the pillars delineates IA into overt phases, namely: intussusceptive microvascular growth, intussusceptive arborization, and intussusceptive branching remodeling. Intussusceptive microvascular growth circumscribes the process of initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced. In contrast, intussusceptive arborization entails formation of serried pillars that remodel the disorganized vascular meshwork into the typical tree-like arrangement. Optimization of local vascular branching geometry occurs through intussusceptive branching remodeling so that the vasculature is remodeled to meet the local demand. In addition, IA is important in creation of the local organ-specific angioarchitecture. While hemodynamic forces have proven direct effects on IA, with increase in blood flow resulting in initiation of pillars, the preponderant mechanisms are unclear. Molecular control of IA has so far not been unequivocally elucidated but interplay among several factors is probably involved. Future investigations are strongly encouraged to focus on interactions among angiogenic growth factors, angiopoetins, and related receptors.

Coronary optical frequency domain imaging (OFDI) for in vivo evaluation of stent healing: comparison with light and electron microscopy

Aims Coronary late stent thrombosis, a rare but devastating complication, remains an important concern in particular with the increasing use of drug-eluting stents. Notably, pathological studies have indicated that the proportion of uncovered coronary stent struts represents the best morphometric predictor of late stent thrombosis. Intracoronary optical frequency domain imaging (OFDI), a novel second-generation optical coherence tomography (OCT)-derived imaging method, may allow rapid imaging for the detection of coronary stent strut coverage with a markedly higher precision when compared with intravascular ultrasound, due to a microscopic resolution (axial ∼10–20 µm), and at a substantially increased speed of image acquisition when compared with first-generation time-domain OCT. However, a histological validation of coronary OFDI for the evaluation of stent strut coverage in vivo is urgently needed. Hence, the present study was designed to evaluate the capacity of coronary OFDI by electron (SEM) and light microscopy (LM) analysis to detect and evaluate stent strut coverage in a porcine model. Methods and results Twenty stents were implanted into 10 pigs and coronary OFDI was performed after 1, 3, 10, 14, and 28 days. Neointimal thickness as detected by OFDI correlated closely with neointimal thickness as measured by LM (r = 0.90, P < 0.01). The comparison of stent strut coverage as detected by OFDI and SEM analysis revealed an excellent agreement (r = 0.96, P < 0.01). In particular, stents completely covered by OFDI analysis were also completely covered by SEM analysis. All incompletely covered stents by OFDI were also incompletely covered by SEM. Analyses of fibrin-covered stent struts suggested that these may rarely be detected as uncovered stent struts by OFDI. Importantly, optical density measurements revealed a significant difference between fibrin- and neointima-covered coronary stent struts [0.395 (0.35–0.43) vs. 0.53 (0.47–0.57); P < 0.001], suggesting that differences in optical density provide information on the type of stent strut coverage. The sensitivity and specificity for detection of fibrin vs. neointimal coverage was evaluated using receiver-operating characteristic analysis. Conclusion The present study demonstrates that OFDI is a highly promising tool for accurate evaluation of coronary stent strut coverage, as supported by a high agreement between OFDI and light and electron microscopic analysis. Furthermore, our data indicate that optical density measurements can provide additional information with respect to the type of stent strut coverage, i.e. fibrin vs. neointimal coverage. Therefore, coronary OFDI analysis will provide important information on the biocompatibility of coronary stents.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Tumor Recovery by Angiogenic Switch from Sprouting to Intussusceptive Angiogenesis after Treatment with PTK787/ZK222584 or Ionizing Radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Hypoxia-inducible factor 1 alpha in high-risk breast cancer: an independent prognostic parameter?

BackgroundHypoxia-inducible factor 1 alpha (hif-1α) furnishes tumor cells with the means of adapting to stress parameters like tumor hypoxia and promotes critical steps in tumor progression and aggressiveness. We investigated the role of hif-1α expression in patients with node-positive breast cancer.MethodsTumor samples from 77 patients were available for immunohistochemistry. The impact of hif-1α immunoreactivity on survival endpoints was determined by univariate and multivariate analyses, and correlations to clinicopathological characteristics were determined by cross-tabulations.Resultshif-1α was expressed in 56% (n = 43/77) of the patients. Its expression correlated with progesterone receptor negativity (P = 0.002). The Kaplan–Meier curves revealed significantly shorter distant metastasis-free survival (DMFS) (P = 0.04, log-rank) and disease-free survival (DFS) (P = 0.04, log-rank) in patients with increased hif-1α expression. The difference in overall survival (OS) did not attain statistical significance (5-year OS, 66% without hif-1α expression and 55% with hif-1α expression; P = 0.21). The multivariate analysis failed to reveal an independent prognostic value for hif-1α expression in the whole patient group. The only significant parameter for all endpoints was the T stage (T3/T4 versus T1/T2: DMFS, relative risk = 3.16, P = 0.01; DFS, relative risk = 2.57, P = 0.03; OS, relative risk = 3.03, P = 0.03). Restricting the univariate and multivariate analyses to T1/T2 tumors, hif-1α expression was a significant parameter for DFS and DMFS.Conclusionshif-1α is expressed in the majority of patients with node-positive breast cancer. It can serve as a prognostic marker for an unfavorable outcome in those with T1/T2 tumors and positive axillary lymph nodes.

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart.

Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain‐ and loss‐of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor‐B (VEGF‐B) in the heart. A cardiomyocyte‐specific VEGF‐B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia‐reperfusion. VEGF‐B increased VEGF signals via VEGF receptor‐2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF‐B transgenic, gene‐targeted or wildtype rats. Importantly, we also show that VEGF‐B expression is reduced in human heart disease. Our data indicate that VEGF‐B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Disruption of Notch1 induces vascular remodeling, intussusceptive angiogenesis, and angiosarcomas in livers of mice

BACKGROUND & AIMS Notch signaling mediates embryonic vascular development and normal vascular remodeling; Notch1 knockout mice develop nodular regenerative hyperplasia (NRH). The pathogenesis of NRH is unclear, but has been associated with vascular injury in the liver sinusoids in clinical studies. We investigated the role of Notch1 signaling in liver sinusoidal endothelial cells (LSECs). METHODS We studied MxCre Notch1(lox/lox) mice (conditional knockout mice without tissue-specific disruption of Notch1); mice with hepatocyte-specific knockout were created by crossing Notch1(lox/lox) with AlbCre(+/-) mice. Portal vein pressure was measured; morphology of the hepatic vasculature was assessed by histologic and scanning electron microscopy analyses. We performed functional and expression analyses of isolated liver cells. RESULTS MxCre-induced knockout of Notch1 led to NRH, in the absence of fibrosis, with a persistent increase in proliferation of LSECs. Notch1 deletion led to de-differentiation, vascular remodeling of the hepatic sinusoidal microvasculature, intussusceptive angiogenesis, and dysregulation of ephrinB2/EphB4 and endothelial tyrosine kinase. Time-course experiments revealed that vascular changes preceded node transformation. MxCre Notch1(lox/lox) mice had reduced endothelial fenestrae and developed portal hypertension and hepatic angiosarcoma over time. In contrast, mice with hepatocyte-specific disruption of Notch1 had a normal phenotype. CONCLUSIONS Notch1 signaling is required for vascular homeostasis of hepatic sinusoids; it maintains quiescence and differentiation of LSECs in adult mice. Disruption of Notch1 signaling in LSECs leads to spontaneous formation of angiosarcoma, indicating its role as a tumor suppressor in the liver endothelium.

Intussusceptive angiogenesis: pillars against the blood flow.

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well‐known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.

Everolimus Augments the Effects of Sorafenib in a Syngeneic Orthotopic Model of Hepatocellular Carcinoma

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib. Mol Cancer Ther; 10(6); 1007–17. ©2011 AACR.

NADPH oxidase-independent formation of extracellular DNA traps by basophils

Basophils are primarily associated with a proinflammatory and immunoregulatory role in allergic diseases and parasitic infections. Recent studies have shown that basophils can also bind various bacteria both in the presence and the absence of opsonizing Abs. In this report, we show that both human and mouse basophils are able to produce mitochondrial reactive oxygen species and to form extracellular DNA traps upon IL-3 priming and subsequent activation of the complement factor 5 a receptor or FcεRI. Such basophil extracellular traps (BETs) contain mitochondrial, but not nuclear DNA, as well as the granule proteins basogranulin and mouse mast cell protease 8. BET formation occurs despite the absence of any functional NADPH oxidase in basophils. BETs can be found in both human and mouse inflamed tissues, suggesting that they also play a role under in vivo inflammatory conditions. Taken together, these findings suggest that basophils exert direct innate immune effector functions in the extracellular space.