Expression Of Angiogenic Growth Factors Research Articles

Expression of angiogenic growth factors VEGF, bFGF and ANG1 in colon cancer after bevacizumab treatment in vitro: A potential self-regulating mechanism.

The present study aimed to examine changes in the expression of angiogenic growth factors in vascular endothelial cells isolated from colon cancer after bevacizumab treatment invitro, and to explore a potential mechanism of their self-regulation as a possible mechanism for antiangiogenic therapy failure in clinics. Vascular endothelial cells were isolated from tumors of colon cancer patients and transfected with recombinant adeno-associated virus type2-vascular endothelial growth factor (VEGF) or pGIPZ-VEGF RNA interference in order to upregulate or downregulate VEGF expression. Changes in VEGF expression and its correlation with the expression of angiogenesis-related factors, including basic fibroblast growth factor (bFGF) and angiopoietin1 (ANG1), after treatment with bevacizumab invitro, were investigated. The results showed that in cells with VEGF overexpression, bFGF and ANG1 were downregulated, whereas in cells in which VEGF was knocked down, upregulation of bFGF and ANG1 was detected. In cells treated with bevacizumab, a significant upregulation of VEGF and downregulation of bFGF and ANG1 were observed. Our data indicate that after bevacizumab treatment, a potential self-regulating mechanism of angiogenic growth factors in colon cancer-derived endothelial cells is activated, which may explain why current antiangiogenic therapy with bevacizumab has limited effects in prolonging the survival of colon cancer patients.

Mechanical strain stimulates vasculogenesis and expression of angiogenesis guidance molecules of embryonic stem cells through elevation of intracellular calcium, reactive oxygen species and nitric oxide generation

ObjectivesDifferentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. Methods and resultsTreatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. ConclusionsMechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB.

EPCs enhance angiogenesis in renal regeneration.

Decellularized renal scaffolds have previously been used for renal regeneration following partial nephrectomy, in which angiogenesis played a key role. In this study, rats underwent partial nephrectomy and repaired with decellularized renal scaffolds. Subsequently, the labeled EPCs were intravenously injected into rats in EPCs group, and the control group received an equal amount of phosphate-buffer saline (PBS). We chose 1, 2 and 4 weeks post operation as time point. Average microvascular density (aMVD) analyses revealed higher angiogenesis in EPCs group compared with the control group. The expression of angiogenic growth factors including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and hypoxia-inducible factors 1-alpha (HIF-1α), was generally higher in the EPCs group in all weeks (1, 2 and 4), and peaked in week 2. EPCs were observed to home into renal injury site, promoting angiogenesis across the renal parenchyma-scaffold interface to be potentially used as bridges for EPCs to migrate into the implanted scaffolds. Administration of exogenous EPCs promotes angiogenesis and vasculogenesis in decellularized renal scaffolds-mediated renal regeneration, providing adequate microenvironment for kidney recovery post renal injury.

Angiogenic Peptide Nanofibers Improve Wound Healing in STZ-Induced Diabetic Rats.

Low expressions of angiogenic growth factors delay the healing of diabetic wounds by interfering with the process of blood vessel formation. Heparin mimetic peptide nanofibers can bind to and enhance production and activity of major angiogenic growth factors, including VEGF. In this study, we showed that heparin mimetic peptide nanofibers can serve as angiogenic scaffolds that allow slow release of growth factors and protect them from degradation, providing a new therapeutic way to accelerate healing of diabetic wounds. We treated wounds in STZ-induced diabetic rats with heparin mimetic peptide nanofibers and studied repair of full-thickness diabetic skin wounds. Wound recovery was quantified by analyses of re-epithelialization, granulation tissue formation and blood vessel density, as well as VEGF and inflammatory response measurements. Wound closure and granulation tissue formation were found to be significantly accelerated in heparin mimetic gel treated groups. In addition, blood vessel counts and the expressions of alpha smooth muscle actin and VEGF were significantly higher in bioactive gel treated animals. These results strongly suggest that angiogenic heparin mimetic nanofiber therapy can be used to support the impaired healing process in diabetic wounds.

The Hidden Side of Disodium Cromolyn: from Mast Cell Stabilizer to an Angiogenic Factor and Antitumor Agent.

Scattered data suggested that disodium cromolyn, well known as a mast cell stabilizer shows some effects on tumor cells and tumor-associated newly formed vascular networks. Most of these studies used tumor cell lines assessed by in vitro studies. Nor disodium cromolyn effects on melanoma cell lines were studied yet, neither its influence on recruited tumor blood vessels or angiogenic growth factors expression. We designed here a study regarding disodium cromolyn effects on A375 melanoma tumor cells implanted on chick embryo chorioallantoic membrane (CAM) and on blood vessels recruited by the experimental melanoma in the absence of mast cells, knowing that within CAM, the existence of mast cells are not certified yet. We also assessed the role of disodium cromolyn on the expression of several angiogenic growth factors. Disodium cromoglycate differentially acts on tumor cells and blood vessels. Extensive necrotic areas of experimental melanoma together with an increased number of peritumor blood vessels were observed in treated specimens as compared with untreated tumors. Disodium cromolyn inhibited VEGF and PDGF-BB expression, and had no effects on EG VEGF expression between treated and non treated specimens in a mast cells free microenvironment. Our results sustain the direct antitumor effects of sodium cromolyn and suggest the involvement of several growth factors in the recruitment of tumor vessels by A375 melanoma tumor cells. The expression of growth factors is differentially influenced by sodium cromolyn treatment.

Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor.

BackgroundCritical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno‐associated virus (AAV) and lentivirus have been tested in preclinical models.Methods and ResultsWe present preclinical results of ischemia (hypoxia)‐regulated conditionally silenced (CS) AAV–human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV‐CS‐hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra‐arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV‐CS‐hVEGF. AAV‐CS‐hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch,SDF1, Angiopoietin, and Ephrin‐B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects.ConclusionsRestriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Adipose tissue extract promotes adipose tissue regeneration in an adipose tissue engineering chamber model

An adipose tissue engineering chamber model of spontaneous adipose tissue generation from an existing fat flap has been described. However, the chamber does not completely fill with adipose tissue in this model. Here, the effect of adipose tissue extract (ATE) on adipose tissue regeneration was investigated. In vitro, the adipogenic and angiogenic capacities of ATE were evaluated using Oil Red O and tube formation assays on adipose-derived stem cells (ASCs) and rat aortic endothelial cells (RAECs), respectively. In vivo, saline or ATE was injected into the adipose tissue engineering chamber 1week after its implantation. At different time points post-injection, the contents were morphometrically, histologically, and immunohistochemically evaluated, and the expression of growth factors and adipogenic genes was analyzed by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. With the exception of the baseline control group, in which fat flaps were not inserted into a chamber, the total volume of fat flap tissue increased significantly in all groups, especially in the ATE group. Better morphology and structure, a thinner capsule, and more vessels were observed in the ATE group than in the control group. Expression of angiogenic growth factors and adipogenic markers were significantly higher in the ATE group. ATE therefore significantly promoted adipose tissue regeneration and reduced capsule formation in an adipose tissue engineering chamber model. These data suggest that ATE provides a more angiogenic and adipogenic microenvironment for adipose tissue formation by releasing various cytokines and growth factors that also inhibit capsule formation.

Abstract B30: MicoRNA mediated regulation of antiangiogenic resistance in glioblastoma

Abstract Anti-angiogeneic therapies are being used in glioma due to high expression of vascular and angiogenic growth factors but without improved survival and tumor recur. Our study used vatalanib (PTK) in U251 tumor cells which secretes proangiogenic and proinvasive proteins. We found 7 proteins (MMP-2, MMP-9, SDF-1α, Tie-2, MCP-1, IL-8, VE Cadherin, and VEGFA) which were altered greater than 20 fold in both U251 complete medium and lysate. We hypothesized that miRNA regulation may be involved in overexpression of these secreted factors that might be responsible for antiangiogenic resistance in glioma. We performed miRNA profiling (Affymetrix miRNA v3 platform) in U251 glioma cell line treated with or without PTK drug under hypoxia and normoxia conditions. Fold changes were estimated using normoxia group as control against normoxia+PTK, hypoxia, hypoxia+PTK, and changes greater than 5 fold were considered for further analysis to investigate potential targets. We found miR-15a and miR-21 were > 7-fold upregulated in group with hypoxia+PTK treatment. Targetscan analysis showed mir-15a may target TNFAIP3, VEGFA and mir21 can target MMP-2, which were altered in U251 cell lysate. Our present study identified novel microRNAs and gene targets which may lead to antiangiogenic resistance in glioma and provide a basis for future clinical trials. In future, functional assays will confirm the role of candidate miRNAs in tumor vasculature development following antiangiogenic drug treatment. Citation Format: Meenu Jain, Bhagelu Achyut, Adarsh Shankar, Kartik Angara, Asm Iskander, Ali S. Arbab. MicoRNA mediated regulation of antiangiogenic resistance in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B30.

Intranasal Delivery of Apelin-13 Is Neuroprotective and Promotes Angiogenesis After Ischemic Stroke in Mice.

Apelin is a peptide originally isolated from bovine stomach tissue extracts and identified as an endogenous ligand of the APJ receptor; recent work showed that apelin ameliorates the ischemic injury in the heart and the brain. Being an analogue to the angiotensin II receptor, the apelin/APJ signaling may mediate angiogenesis process. We explored the noninvasive intranasal brain delivery method and investigated therapeutic effects of apelin-13 in a focal ischemic stroke model of mice. Intranasal administration of apelin-13 (4 mg/kg) was given 30 min after the onset of stroke and repeated once daily. Three days after stroke, mice received apelin-13 had significantly reduced infarct volume and less neuronal death in the penumbra. Western blot analyses showed upregulated levels of apelin, apelin receptor APLNR, and Bcl-2 and decreased caspase-3 activation in the apelin-13-treated brain. The proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1β, and chemokine monocyte chemoattractant protein-1 mRNA increased in the ischemic brain, which were significantly attenuated by apelin-13. Apelin-13 remarkably reduced microglia recruitment and activation in the penumbra according to morphological features of Iba-1-positive cells 3 days after ischemia. Apelin-13 significantly increased the expression of angiogenic factor vascular endothelial growth factor and matrix metalloproteinase-9 14 days after stroke. Angiogenesis illustrated by collagen IV + /5-bromo-2′-deoxyuridin + colabeled cells was significantly increased by the apelin-13 treatment 21 days after stroke. Finally, apelin-13 promoted the local cerebral blood flow restoration and long-term functional recovery. This study demonstrates a noninvasive intranasal delivery of apelin-13 after stroke, suggesting that the reduced inflammatory activities, decreased cell death, and increased angiogenesis contribute to the therapeutic benefits of apelin-13.

Mesenchymal–endothelial transition in juvenile angiofibroma?

Conclusion: Mesenchymal–endothelial transition is proposed for juvenile angiofibromas (JAs). Propranolol might be an interesting new medical option in JA treatment, as it reduces mesenchymal cell growth and decreases the number of CD31-positive cells in vitro. Objective: Juvenile angiofibromas (JAs) are rare fibro-vascular tumors affecting almost exclusively adolescent males. Based on morphological aspects of irregularly configured vascular spaces and clinical features, JAs have been proposed to represent a vascular malformation. In general, mesenchymal–endothelial transition has been noted as an important process in tumorigenesis as well as in embryonal development. Methods: The study analyzed effects of vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF) and propranolol on endothelial differentiation (CD31+) of cultured JA cells and their expression of angiogenic growth factors using aortic ring assay and enzyme-linked immunosorbent assay. Results: Following VEGF/bFGF supplement to cultured mesenchymal cells, an average of 4.47% (±2.35%) CD31-positive cells were found (p < 0.001). Propranolol addition reduced the number of CD31-positive cells and inhibited mesenchymal cell growth. The aortic ring assay and ELISA investigation indicated no increased angiogenic growth factor secretion from cultured JA mesenchymal cells.

Effects of pneumoperitoneum preconditioning on endothelial progenitor cells and renal protective mechanism in rats

To explore the pneumoperitoneum-mediated renoprotective effects of preconditioning, mobilizing and homing of endothelial progenitor cells (EPCs) in rats. A total of 40 rats were randomized by a numerical table into 5 groups of gasless (C), pneumoperitoneum injury (Pp), long-term pneumoperitoneum preconditioning (P-L), mid-term pneumoperitoneum preconditioning (P-M) and short-term pneumoperitoneum preconditioning (P-S). C group had a pneumoperitoneum pressure of 0 mmHg; Pp group 15 mmHg, time 60 min; P-L, P-M, P-S groups were deflated and deflated preconditioning before pneumoperitoneum, then the same as Pp group, P-L group: inflation time was 25 min, gas discharge time 10 min; P-M group: 15 min, 10 min; P-S group: 5 min, 10 min. At 24 h post-operation, the animals were sacrificed by destroying cervical spine. And the specimens of venous blood and kidneys were harvested. Also the extent of renal injury, the homing of EPCs, the proliferation and angiogenesis of renal endothelial cell and the expression of angiogenic growth factor were analyzed. Compared with Pp group, P-L, P-M and P-S groups exhibited significant improvements in renal function, morphology and histological score (1.88 ± 0.35, 1.63 ± 0.52, 1.75 ± 0.46 vs 2.38 ± 0.52, all P < 0.05). The histological scores of P-M and P-S groups improved significantly versus P-L group (both P < 0.05). P-M and P-S groups showed no significant difference in histological score (P > 0.05). The number of EPCs in kidneys increased in P-L, P-M and P-S groups versus Pp group (2.18% ± 0.14%, 2.87% ± 0.29%, 2.90% ± 0.24% vs 1.73% ± 0.19%, all P < 0.05). The EPCs numbers of P-M and P-S groups were more than that of P-L group (both P < 0.05). And no significant difference existed between P-M and P-S groups (P > 0.05). Compared with Pp group, EPCs of P-L, P-M and P-S groups markedly increased in kidneys. No significant difference existed between P-M and P-S groups, but P-L group was the lowest. Also there was an up-regulated expression of stromal cell derived factor 1-α in pretreated kidneys versus Pp group (all P < 0.05). And P-M and P-S groups increased markedly. Pneumoperitoneum-mediated preconditioning protects against kidney injury by promoting EPC homing and enhancing endothelial cell and vascular proliferations. And short and medium-term preconditioning protocols are more effective for protecting kidneys.

Graphene Potentiates the Myocardial Repair Efficacy of Mesenchymal Stem Cells by Stimulating the Expression of Angiogenic Growth Factors and Gap Junction Protein

Stem cell therapy has emerged as a potential modality for myocardial infarction treatment. Mesenchymal stem cells (MSCs) exert reparative actions in the injured myocardium mainly through the secretion of paracrine factors. In addition, the overexpression of connexin 43 (Cx43), a gap junction protein, promotes cardiac repair and function restoration. It is known that MSCs in a spheroid form, which have enhanced cell–cell interaction, exhibit enhanced expression of paracrine factors and Cx43. However, cell‐extracellular matrix (ECM) interactions, which also contribute to growth factor expression, are very limited in MSC spheroids. Reduced graphene oxide (RGO) shows high affinity toward ECM proteins, such as fibronectin (FN), and high electrical conductivity. In this study, by incorporating FN‐adsorbed RGO flakes into MSC spheroids, it is possible to enhance the cell–ECM interactions and, subsequently, the paracrine factor expression in the MSCs in spheroids. Cx43 is also upregulated likely due to the enhanced paracrine factor expression and electrical conductivity of RGO. The injection of MSC–RGO hybrid spheroids into the infarcted hearts enhances cardiac repair compared with the injection of RGO flakes or MSC spheroids. This study demonstrates that RGO can effectively improve the therapeutic efficacy of MSCs for ischemic heart diseases.

Suppression of In Vivo Neovascularization by the Loss of TRPV1 in Mouse Cornea.

To investigate the effects of loss of transient receptor potential vanilloid receptor 1 (TRPV1) on the development of neovascularization in corneal stroma in mice. Blocking TRPV1 receptor did not affect VEGF-dependent neovascularization in cell culture. Lacking TRPV1 inhibited neovascularization in corneal stroma following cauterization. Immunohistochemistry showed that immunoreactivity for active form of TGFβ1 and VEGF was detected in subepithelial stroma at the site of cauterization in both genotypes of mice, but the immunoreactivity seemed less marked in mice lacking TRPV1. mRNA expression of VEGF and TGFβ1 in a mouse cornea was suppressed by the loss of TRPV1. TRPV1 gene ablation did not affect invasion of neutrophils and macrophage in a cauterized mouse cornea. Blocking TRPV1 signal does not affect angiogenic effects by HUVECs in vitro. TRPV1 signal is, however, involved in expression of angiogenic growth factors in a cauterized mouse cornea and is required for neovascularization in the corneal stroma in vivo.

PS-211 Early Postnatal Systemic Lipopolysaccharide Increases Pro-inflammatory Cytokines And Angiogenic Growth Factors In The Lungs And Leads To The Phenotype Of New Bpd In Neonatal Rats

Background Recently bronchopulmonary dysplasia (BPD) occurs not infrequently without an exposure to hyperoxia and/or mechanical ventilation in very preterm infants. To simulate the recent pattern of BPD, novel animal model of BPD induced by inflammation alone is needed. We were to establish a rat model of BPD induced by postnatal systemic lipopolysaccharide (LPS) administration alone. Methods Two days before delivery, 1 μg of LPS or vehicle (V) was injected into each amniotic sac, and after birth 0.25 mg/kg of LPS or V was injected into peritoneum of pups at P1, P3, and P5. At P7 and P14, bronchoalveolar lavage (BAL) and lung harvest were performed. BAL fluid (BALF) and peripheral blood (PB) were examined for blood cell counts and lung tissue was examined for morphometry, vascular density, neutrophil infiltration, and expressions of pro-inflammatory cytokines and angiogenic growth factors. Results Postnatal systemic LPS significantly increased the neutrophil counts in PB, BALF and within the alveoli and expressions of pro-inflammatory cytokines and angiogenic growth factors at P7. When postnatal systemic LPS was preceded by intra-amniotic LPS administration, these findings were not observed. Postnatal systemic LPS led to significant disruption of alveolar and pulmonary vascular developments at P14. Conclusions Early postnatal systemic LPS induced systemic and pulmonary pro-inflammatory responses and disrupted alveolar and pulmonary vascular developments. This rat model of new BPD induced by early postnatal inflammation per se without an exposure to hyperoxia can be used to test the effects of anti-inflammatory agents for BPD.

Angiogenic growth factor expression in benign and malignant vascular tumours

Angiosarcomas are rare malignant vascular tumours. Angiosarcoma expression of vascular endothelial growth factor (VEGF) has previously been reported, but angiosarcoma expression of other angiogenic growth factors has not been systematically studied. Non-VEGF angiogenic growth factors are a potential mechanism of resistance to VEGF-targeted therapy, but they also represent potential therapeutic targets.Immunohistochemistry analysis evaluated the expression of 13 angiogenic growth factors and receptors in 27 separate benign and malignant archived human vascular tumour samples. The expression of 55 angiogenesis-related proteins was subsequently profiled in five fresh human angiosarcoma tumour samples using antibody arrays.Angiosarcomas expressed a variety of angiogenic growth factors. Significantly higher levels of Notch1 were detected compared with benign haemangiomas (p=0.033), but lower levels of basic fibroblast growth factor (bFGF) compared to benign haemangiomas (p=0.07) and inflammatory vascular lesions (p=0.009). Vascular tumour expression of FGF receptor (FGFR)-1 correlated with angiopoietin (Ang)-2, Tie2, hepatocyte growth factor (HGF) and Notch1 expression (p=0.001, p=0.001, p<0.001 and p<0.001 respectively). Notch1 also correlated with Tie2 expression (p=0.004).In conclusion, angiosarcomas express multiple angiogenic growth factors. Treatments could be targeted at individual angiogenic growth factors. However, our findings provide a rationale for combination therapy, or for treatments that target common downstream signalling intermediaries, such as Akt, mTOR or ERK.

Hypoxia-inducible factor-1α gene polymorphisms in early and late onset preeclampsia in Sinhalese women

IntroductionEarly (EPE) and late (LPE) onset preeclampsia are increasingly being recognized as two distinct disorders. Placental vascular defects are more common in EPE. Hypoxia Inducible Factor 1α (HIF1α) regulates the expression of many angiogenic growth factors in the placenta. We studied the association of two polymorphisms in the HIF1α gene (rs11549465 and rs10873142) with EPE and LPE. Methods175 nulliparous Sinhalese women with preeclampsia and 171 normotensive women matched for age, ethnicity, parity and BMI were recruited at two tertiary care hospitals in Colombo. Preeclampsia was diagnosed using international guidelines. DNA extracted from peripheral blood was genotyped using Sequenom MassARRAY. ResultsHIF1α rs11549465 dominant model and T allele were reduced in women who developed EPE compared to controls [P = 0.002, OR (95% CI) = 0.3 (0.1–0.7)], in preeclamptic women who delivered small for gestational age babies [P = 0.02, OR (95% CI) = 0.5 (0.2–0.9)] compared to controls and in women who developed EPE compared to those who developed LPE [P = 0.006, OR (95% CI) = 0.3 (0.1–0.7)]. ConclusionOur results demonstrate a protective effect of the T allele in LPE and normal pregnancy, which is relatively lacking in EPE due to low prevalence of this protective allele. HIF1α rs11549465 T allele was previously demonstrated to be associated with a higher transcriptional activity and increased angiogenesis. Inherited susceptibility to increased HIF1α expression resulting in the up-regulation of angiogenic genes may mediate a protective effect in normal pregnancy and pregnancy complicated by LPE.

Epidermal or Dermal Specific Knockout of PHD-2 Enhances Wound Healing and Minimizes Ischemic Injury

IntroductionHypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.MethodsEpidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.ResultsqRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).ConclusionsPHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.

Anti-Parstatin Promotes Angiogenesis and Ameliorates Left Ventricular Dysfunction during Pressure Overload

Parstatin, a novel protease activated receptor-1 (PAR-1) derived peptide is a potent inhibitor of angiogenesis. We and others have reported that imbalance between angiogenic growth factors and anti-angiogenic factors results in transition from compensatory cardiac hypertrophy to heart failure in a pressure overload condition. Though cardio protective role of parstatin was shown previously in ischemic cardiac injury, its role in pressure overload cardiac injury is yet to unveil. We hypothesize that supplementing anti-parstatin antibody during pressure overload condition augments angiogenesis and ameliorate left ventricular dysfunction and heart failure. To verify this, we created ascending aortic banding in mice to mimic pressure overload condition and then treated mice with anti-parstatin antibody. Left ventricular function was assessed by echocardiography and pressure-volume loop study. Angiogenic growth factors and anti-angiogenic factors along with MMP-2,-9 were evaluated by western blot and immunohistochemistry. Results: our results showed an improved left ventricular function in anti-parstatin treated aortic banding hearts compared to their corresponding wild type controls. Expression of angiogenic growth factor, VEGF, MMP-2 and CD31 expression was increased in treated aortic banding hearts compared to their corresponding wild type controls. Our results suggest that treating pressure overload mice with anti-parstatin antibody augments angiogenesis and ameliorates left ventricular dysfunction.

Wnt/β-catenin signaling pathway may regulate the expression of angiogenic growth factors in hepatocellular carcinoma

The Wnt/β-catenin signaling pathway plays a key role during hepatocellular carcinoma (HCC) genesis and development. The present study aimed to investigate the effects of the Wnt/β-catenin signaling pathway on the expression of angiogenic growth factors involved in HCC. The HCC HepG2 cell line was transfected with small interfering RNA (siRNA) against β-catenin. After 72 and 96 h, protein was extracted and the expression levels of β-catenin, matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF)-A, VEGF-C and basic fibroblast growth factor (bFGF) were detected by western blot analysis. β-catenin protein expression was inhibited at both time points. Notably, MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF protein expression levels decreased at 72 h and then increased at 96 h after transfection. Our results demonstrated that in HCC cells, the Wnt/β-catenin signaling pathway may regulate the protein expression of the angiogenic factors, MMP-2, MMP-9, VEGF-A, VEGF-C and bFGF. These proteins were downstream of β-catenin signaling and were also regulated by other factors. In conclusion, the Wnt/β-catenin signaling pathway may contribute to the regulation of HCC angiogenesis, infiltration and metastasis through regulating the expression of these angiogenic factors.

ITE and TCDD Differentially Regulate the Vascular Remodeling of Rat Placenta via the Activation of AhR

Vascular remodeling in the placenta is essential for normal fetal development. The previous studies have demonstrated that in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an environmental toxicant) induces the intrauterine fetal death in many species via the activation of aryl hydrocarbon receptor (AhR). In the current study, we compared the effects of 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and TCDD on the vascular remodeling of rat placentas. Pregnant rats on gestational day (GD) 15 were randomly assigned into 5 groups, and were exposed to a single dose of 1.6 and 8.0 mg/kg body weight (bw) ITE, 1.6 and 8.0 µg/kg bw TCDD, or an equivalent volume of the vehicle, respectively. The dams were sacrificed on GD20 and the placental tissues were gathered. The intrauterine fetal death was observed only in 8.0 µg/kg bw TCDD-exposed group and no significant difference was seen in either the placental weight or the fetal weight among all these groups. The immunohistochemical and histological analyses revealed that as compared with the vehicle-control, TCDD, but not ITE, suppressed the placental vascular remodeling, including reduced the ratio of the placental labyrinth zone to the basal zone thickness (at least 0.71 fold of control), inhibited the maternal sinusoids dilation and thickened the trophoblastic septa. However, no marked difference was observed in the density of fetal capillaries in the labyrinth zone among these groups, although significant differences were detected in the expression of angiogenic growth factors between ITE and TCDD-exposed groups, especially Angiopoietin-2 (Ang-2), Endoglin, Interferon-γ (IFN-γ) and placenta growth factor (PIGF). These results suggest ITE and TCDD differentially regulate the vascular remodeling of rat placentas, as well as the expression of angiogenic factors and their receptors, which in turn may alter the blood flow in the late gestation and partially resulted in intrauterine fetal death.