Luisa Iruela-Arispe

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Interfere With Angiogenesis by Inhibiting the Geranylgeranylation of RhoA

Angiogenesis is implicated in the pathogenesis of cancer, rheumatoid arthritis, and atherosclerosis and in the treatment of coronary artery and peripheral vascular disease. Here, cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are shown to interfere with angiogenesis. In vivo, the HMG-CoA reductase inhibitor simvastatin dose-dependently inhibited capillary growth in both vascular endothelial growth factor–stimulated chick chorioallantoic membranes and basic fibroblast growth factor–stimulated mouse corneas. In vitro, the development of tubelike structures by human microvascular endothelial cells cultured on 3D collagen gels was inhibited at simvastatin concentrations similar to those found in the serum of patients on therapeutic doses of this agent. HMG-CoA reductase inhibitors interfered with angiogenesis via inhibition of the geranylgeranylation and membrane localization of RhoA. Simvastatin inhibited membrane localization of RhoA with a concentration dependence similar to that for the inhibition of tube formation, whereas geranylgeranyl pyrophosphate, the substrate for the geranylgeranylation of Rho, reversed the effect of simvastatin on tube formation and on the membrane localization of RhoA. Furthermore, tube formation was inhibited by GGTI, a specific inhibitor of the geranylgeranylation of Rho; by C3 exotoxin, which inactivates Rho; and by the adenoviral expression of a dominant-negative RhoA mutant. The expression of a dominant-activating RhoA mutant reversed the effect of simvastatin on tube formation. Finally, HMG-CoA reductase inhibitors inhibited signaling by vascular endothelial growth factor, Akt, and focal adhesion kinase, three RhoA-dependent pathways known to be involved in angiogenesis. This study demonstrates a new relationship between lipid metabolism and angiogenesis and an antiangiogenic effect of HMG-CoA reductase inhibitors with possible important therapeutic implications.

Thrombospondin‐1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice

The function of the endogenous angiogenesis inhibitor thrombospondin‐1 (TSP‐1) in tissue repair has remained controversial. We established transgenic mice with targeted overexpression of TSP‐1 in the skin, using a keratin 14 expression cassette. TSP‐1 transgenic mice were healthy and fertile, and did not show any major abnormalities of normal skin vascularity, cutaneous vascular architecture, or microvascular permeability. However, healing of full‐thickness skin wounds was greatly delayed in TSP‐1 transgenic mice and was associated with reduced granulation tissue formation and highly diminished wound angiogenesis. Moreover, TSP‐1 potently inhibited fibroblast migration in vivo and in vitro. These findings demonstrate that TSP‐1 preferentially interfered with wound healing‐associated angiogenesis, rather than with the angiogenesis associated with normal development and skin homeostasis, and suggest that therapeutic application of angiogenesis inhibitors might potentially be associated with impaired wound vascularization and tissue repair.

Cellular Responses of Bioabsorbable Polymeric Material and Guglielmi Detachable Coil in Experimental Aneurysms

Background and Purpose— Acceleration of healing mechanisms is a promising approach to improve current limitations of endovascular aneurysm therapy with the use of platinum coils. We evaluated a new endovascular therapeutic, bioabsorbable polymeric material (BPM), which may promote cellular reaction in the aneurysms. Methods— Four different concentrations of lactide/glycolic acid copolymer [poly(d-l-lactic-co-glycolic acid)] (PLGA), 85/15, 75/25, 65/35, and 50/50, were used as BPMs. Sixteen experimental aneurysms were created in 8 swine. Eight-millimeter-long spiral-shaped BPMs were surgically implanted in the aneurysms without tight packing (n=3 for each BPM). Guglielmi detachable coils (GDCs) were used as control (n=4). The animals were killed 14 days after embolization, and angiographic, histological, and immunohistochemical analyses were performed. Results— Despite loose packing of aneurysms with BPMs, faster BPMs such as 50/50 or 65/35 PLGA demonstrated more mature collagen formation and fibrosis in the sac and neck of the aneurysm. One aneurysm treated with 65/35 PLGA, 1 treated with 75/25 PLGA, and all 3 treated with 85/15 PLGA showed a neck remnant on angiography. There was a linear relationship between collagen levels and polymer degradation properties (r =−0.9513). Conclusions— This preliminary animal study indicates that acceleration of aneurysm healing with the use of BPM is feasible. This concept can be applied to decrease and perhaps prevent aneurysmal recanalization after endovascular treatment of cerebral aneurysms.

ELF-1 Is a Transcriptional Regulator of the Tie2 Gene During Vascular Development

Abstract — Vascular development requires the tightly coordinated expression of several growth factors and their receptors. Among these are the Tie1 and Tie2 receptors, which are almost exclusively endothelial cell–specific. The critical transcriptional regulators of vascular-specific gene expression remain largely unknown. The Ets factors are a family of evolutionarily conserved transcription factors that regulate genes involved in cellular growth and differentiation. We have recently shown that the Ets factor NERF is a strong transactivator of the Tie1 and Tie2 genes. To extend these studies, we have begun to identify the Ets factors that are expressed in developing blood vessels of the chicken chorioallantoic membrane (CAM), a highly vascular embryonic network. RNA was extracted from microdissected CAM blood vessels, and reverse transcriptase–polymerase chain reaction was performed using oligonucleotides encoding conserved amino acids within the Ets domain. One of the polymerase chain reaction fragments was subcloned and identified as the chicken homologue of the Ets factor ELF-1, cELF-1. ELF-1 is most closely related to the Ets factor NERF. In situ hybridization and immunohistochemistry demonstrate that cELF-1 is enriched in developing chicken blood vessels. cELF-1 is also a strong transactivator of the Tie1 and Tie2 genes and can bind to conserved Ets sites within the promoters of these genes. A complex of similar size forms when gel shifts are performed with cellular extracts derived from the CAM blood vessels, which is recognized by an antibody against cELF-1. In summary, ELF-1 belongs to a subset of Ets factors that regulate vascular-specific gene expression during blood vessel development.

HAART drugs induce mitochondrial damage and intercellular gaps and gp120 causes apoptosis.

HIV-1 infection is associated with serious cardiovascular complications, but the roles of HIV-1, viral proteins, and highly active antiretroviral therapy (HAART) drugs are not understood. HAART decreases the overall risk of heart disease but leads to metabolic disturbances and possibly coronary artery disease. We investigated toxicities of HIV-1, HIV-1 glycoprotein 120 (gp 120), and HAART drugs for human coronary artery endothelial cells (CAECs), brain microvascular endothelial cells, and neonatal rat ventricular myocytes (NRVMs). HIV-1 and gp 120, but not azidothymidine (AZT), induced apoptosis of NRVMs and CAECs. Ethylisothiourea, an inhibitor of nitric oxide synthase, inhibited apoptosis induction by gp 120. AZT, HIV-1, and gp 120 all damaged mitochondria of cardiomyocytes. HAART drugs, AZT, and indinavir, but not HIV-1, produced intercellular gaps between confluent endothelial cells and decreased transendothelial electrical resistance. In conclusion, HIV-1 and gp 120 induce toxicity through induction of cardiomyocyte and endothelial cell apoptosis. HAART drugs disrupt endothelial cell junctions and mitochondria and could cause vascular damage.

Increased Expression of A Disintegrin and Metalloproteinase Thrombospondin–1 in Thrombosed Hemodialysis Grafts

PURPOSE To use proteomic analysis to identify upregulated and downregulated proteins in thrombosed hemodialysis graft specimens. One of these significantly upregulated proteins was a disintegrin and metalloproteinase thrombospondin-1 (ADAMTS-1), and its expression and activity were determined in thrombosed hemodialysis grafts. MATERIALS AND METHODS Hemodialysis vascular access samples (thrombosed veins, n = 8; control veins, n = 6) were obtained from patients who required surgical revision. Proteomic analysis was performed with isotope-coded affinity tag labeling with multidimensional liquid chromatography followed by tandem mass spectrometry on four thrombosed hemodialysis graft specimens with control veins. Expression of ADAMTS-1 was confirmed by performing immunoprecipitation followed by Western blot analysis. Finally, immunohistochemistry was used to localize expression in a separate group of patients with thrombosed grafts. RESULTS Thirty-nine unique proteins were common to all four patients. ADAMTS-1 was one of the only significantly upregulated protein (>38 fold). ADAMTS-1 expression was confirmed by performing immunoprecipitation and Western blot analysis and was significantly increased. ADAMTS-1 expression was localized to adventitial macrophages and neutrophils of thrombosed grafts. CONCLUSIONS ADAMTS-1 was significantly upregulated in thrombosed hemodialysis grafts by mass spectrometric analysis and Western blot analysis. Expression was localized to adventitial macrophages and leukocytes. It is hypothesized that ADAMTS-1 may be related to intimal hyperplasia in hemodialysis vascular access grafts. Future work is planned on inhibiting ADAMTS-1 expression and determining the effect on intimal hyperplasia in hemodialysis grafts.

Endothelial-Mesenchymal Transition in Vascular Calcification of Ins2Akita/+ Mice.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to normal development and disease processes. Here, we report that EndMTs occur in the diabetic endothelium of Ins2Akita/wt mouse, and show that induction of sex determining region Y-box 2 (Sox2) is a mediator of excess BMP signaling that results in activation of EndMTs and increased vascular calcification. We also find an induction of a complex of serine proteases in the diabetic endothelium, required for the up-regulation of Sox2. Our results suggest that EndMTs contribute to vascular calcification in diabetic arteries.

Cardiac myocyte p38α kinase regulates angiogenesis via myocyte-endothelial cell cross-talk during stress-induced remodeling in the heart

Stress-induced p38 mitogen-activated protein kinase (MAPK) activity is implicated in pathological remodeling in the heart. For example, constitutive p38 MAPK activation in cardiomyocytes induces pathological features, including myocyte hypertrophy, apoptosis, contractile dysfunction, and fetal gene expression. However, the physiological function of cardiomyocyte p38 MAPK activity in beneficial compensatory vascular remodeling is unclear. This report investigated the functional role and the underlying mechanisms of cardiomyocyte p38 MAPK activity in cardiac remodeling induced by chronic stress. Using both in vitro and in vivo model systems, we found that p38 MAPK activity is required for hypoxia-induced pro-angiogenic activity from cardiomyocytes and that p38 MAPK activation in cardiomyocyte is sufficient to promote paracrine signaling-mediated, pro-angiogenic activity. We further demonstrate that VEGF is a paracrine factor responsible for the p38 MAPK-mediated pro-angiogenic activity from cardiomyocytes and that p38 MAPK pathway activation is sufficient for inducing VEGF secretion from cardiomyocytes in an Sp1-dependent manner. More significantly, cardiomyocyte-specific inactivation of p38α in mouse heart impaired compensatory angiogenesis after pressure overload and promoted early onset of heart failure. In summary, p38αMAPK has a critical role in the cross-talk between cardiomyocytes and vasculature by regulating stress-induced VEGF expression and secretion in cardiomyocytes. We conclude that as part of a stress-induced signaling pathway, p38 MAPK activity significantly contributes to both pathological and compensatory remodeling in the heart.

Combined Effects of Bone Morphogenetic Protein 10 and Crossveinless‐2 on Cardiomyocyte Differentiation in Mouse Adipocyte‐Derived Stem Cells

Bone morphogenetic protein (BMP) 10, a cardiac‐restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless‐2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP‐binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte‐like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co‐immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10‐induced proliferation. Differentiation of cardiomyocyte‐like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5‐positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha‐actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube‐colonies and was expressed by endothelial‐like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells.

Abstract A48: M2 macrophage inhibition reverses vascular leaks that cause malignant ascites in late-stage epithelial ovarian cancer

Introduction: Malignant ascites is a common symptom of epithelial ovarian cancer (EOC) that greatly reduces the quality of life for patients and has no good treatment options that target the source of the problem. Our study aims to find the cause of ascites in order for us to better know how to safely reverse this pathology. Experimental Procedures: Murine ID8 EOC tumor cells were implanted intraperitoneally or orthotopically into female C57BL/6 mice, and bioluminescent imaging was used to track the progress of the cancer. GW2580, a very selective colony stimulating factor 1 receptor (CSF1R) inhibitor, was given to mice for two weeks once ascites appeared. Flow cytometry, histology, and endothelial cell permeability assays were used to analyze the effects of blocking macrophages in the model. Flow cytometry and endothelial cell permeability assays were also used to analyze and characterize EOC patient ascites samples. Results: CSF1R inhibition blocked specifically M2 macrophages from the ascites and dramatically reduced ascites volume, leaving mice less anemic. When M2 macrophages numbers were reduced in the ascites, CD8 T cell numbers increased, causing the ascites microenvironment to be less immunosuppressive. Peritoneal vasculature that had grown disorganized and leaky became normalized with treatment. Ascites sera from GW2580-treated mice protected against endothelium permeability, while control mouse sera caused the endothelium to become leaky. This data agrees with our finding that patient ascites sera cause more endothelium permeability if more macrophages were present in the ascites. We also saw macrophage and myeloid cell depletion in the tumor and systemically with GW2580 treatment, along with a consistent reduction in tumor burden. Conclusions: M2 macrophages are key players that cause ascites through deregulating peritoneal vasculature. The leaky vessels are normalized once these macrophages are physically and functionally blocked from the ascites microenvironment. This finding suggests that macrophage inhibition could be a powerful tool for ascites management. VEGF inhibitors are currently in clinical trials for malignant ascites treatment with mixed, sometimes deadly, results. Targeting macrophages, the source of many vessel-deregulating proteins, might make it more difficult for resistance to build up than when just one protein, such as VEGF, is blocked, and CSF1R inhibitors have been shown to be safe and well-tolerated in the clinic. This novel approach toward ascites and EOC management should be further studied. Future work will focus on finding the specific factors that caused the vessel deregulation and normalization, looking at functional differences between macrophage from control and GW2580-treated ascites, seeing if endothelial cell-macrophage direct interaction may have any effects on the vasculature, and performing this study in genetic and human models of EOC. Citation Format: Diana Moughon, Huanhuan He, Shiruyeh Schokrpur, Ziyue Jiang, Madeeha Yaqoob, John David, Luisa Iruela-Arispe, Oliver Dorigo, Lily Wu. M2 macrophage inhibition reverses vascular leaks that cause malignant ascites in late-stage epithelial ovarian cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A48. doi:10.1158/1538-7445.CHTME14-A48