Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis

The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation. CONCLUSION: Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

Angiogenesis in cancer - general pathways and their therapeutic implications

A vast amount of data shows that angiogenesis has a pivotal role in tumor growth, progression, invasiveness and metastasis. This is a complex process involving essential signaling pathways such as vascular endothelial growth factor (VEGF) and Notch in vasculature, as well as additional players such as bone marrow-derived endothelial progenitor cells. Primary tumor cells, stromal cells and cancer stem cells strongly influence vessel growth in tumors. Better understanding of the role of the different pathways and the crosstalk between different cells during tumor angiogenesis are crucial factors for developing more effective anticancer therapies. Targeting angiogenic factors from the VEGF family has become an effective strategy to inhibit tumor growth and so far the most successful results are seen in metastatic colorectal cancer (CRC), renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLL). Despite the initial enthusiasm, the angiogenesis inhibitors showed only moderate survival benefit as monotherapy, along with a high cost and many side effects. Obviously, other important pathways may affect the angiogenic switch, among them Notch signaling pathway attracted a large interest because its ubiquitous role in carcinogenesis and angiogenesis. Herein we present the basics for VEGF and Notch signaling pathways and current advances of targeting them in antiangiogenic, antitumor therapy.

Intussusceptive microvascular growth in tumors

Intussusception is an alternative to the sprouting mode of angiogenesis. The advantage of this mechanism of vascular growth is that blood vessels are generated more rapidly and the capillaries thereby formed are less leaky. This review article summarizes our current knowledge concerning the role played by intussusceptive microvascular growth in tumor growth. Interestingly, an angiogenic switch from sprouting to intussusceptive angiogenesis occurs after treatment with angiogenesis inhibitors and may be considered as a tumor-protective adaptative response.

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.

Src inhibitors in lung cancer: current status and future directions

Src tyrosine kinases regulate multiple genetic and signaling pathways involved in the proliferation, survival, angiogenesis, invasion, and migration of various types of cancer cells They are frequently expressed and activated in many cancer types, including lung cancer. Several Src inhibitors, including dasatinib, saracatinib, bosutinib, and KX2-391, are currently being investigated in clinical trials. Preliminary results of the use of single-agent Src inhibitors in unselected patients with lung cancer show that these inhibitors have a favorable safety profile and anticancer activity. Their combination with cytotoxic chemotherapy, other targeted therapy, and radiation therapy is currently being explored. In this review, we summarize the rationale for and the current status of Src inhibitor development and discuss future directions based on emerging preclinical data.

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.

Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

Impact of MET targeting on tumor-associated angiogenesis and growth of MET mutations-driven models of liver cancer

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

Efficacy of fluoride compounds and stannous chloride as erosion inhibitors in dentine

The aim of this study was to evaluate the anti-erosive effects of different fluoride compounds and one tin compound in the context of the complex pathohistology of dentine erosion, with particular emphasis on the role of the organic portion. Samples were subjected to two experiments including erosive acid attacks (0.05 molar citric acid, pH 2.3; 6 x 2 min/day) and applications (6 x 2 min/day) of the following test solutions: SnCl(2) (815 ppm Sn), NaF (250 ppm F), SnF(2) (250 ppm F, 809 ppm Sn), amine fluoride (AmF, 250 ppm F), AmF/NaF (250 ppm F), and AmF/SnF(2) (250 ppm F, 409 ppm Sn). The demineralised organic fraction was enzymatically removed either at the end of the experiment (experiment 1) or continuously throughout the experiment (experiment 2). Tissue loss was determined profilometrically after 10 experimental days. In experiment 1, the highest erosive tissue loss was found in the control group (erosion only); the AmF- and NaF-containing solutions reduced tissue loss by about 60%, reductions for SnCl(2), AmF/SnF(2), and SnF(2) were 52, 74 and 89%, respectively. In experiment 2, loss values generally were significantly higher, and the differences between the test solutions were much more distinct. Reduction of tissue loss was between 12 and 34% for the AmF- and NaF-containing preparations, and 11, 67 and 78% for SnCl(2), AmF/SnF(2), and SnF(2), respectively. Stannous fluoride-containing solutions revealed promising anti-erosive effects in dentine. The strikingly different outcomes in the two experiments suggest reconsidering current methodologies for investigating anti-erosive strategies in dentine.

Endothelial Progenitor Cells Induce a Phenotype Shift in Differentiated Endothelial Cells towards PDGF/PDGFR Axis-Mediated Angiogenesis

BACKGROUND: Endothelial Progenitor Cells (EPC) support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors. OBJECTIVE: The aim of this study was to investigate the role of PDGF-BB and PDGFR in EPC-mediated angiogenesis of differentiated endothelial cells. METHODS AND RESULTS: Conditioned medium from human EPC (EPC-CM) cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01). EPC-CM increased proliferation (1.39-fold; P<0.001) and migration (2.13-fold; P<0.001) of isolated human umbilical vein endothelial cells (HUVEC), as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01). The capacity of EPC-CM to modulate the PDGFR expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFR (P<0.01). EPC-CM triggered a distinct up-regulation of PDGFR (2.5±0.5; P<0.05) and its phosphorylation (3.6±0.6; P<0.05) in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone. CONCLUSION: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFR , thereby turning the PDGF/PDGFR signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

Bioluminescence imaging of angiogenesis in a murine orthotopic pancreatic cancer model

Angiogenesis is essential for physiological processes as well as for carcinogenesis. New approaches to cancer therapy include targeting angiogenesis. One target is VEGF-A and its receptor VEGFR2. In this study, we sought to investigate pancreatic cancer angiogenesis in a genetically modified VEGFR2-luc-KI mouse.

VEGF-A promotes intussusceptive angiogenesis in the developing chicken chorioallantoic membrane

To assess the impact of vascular endothelial growth factor (VEGF) on intussusceptive angiogenesis.

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.