Potent Angiogenic Growth Factor Research Articles

Vascular Endothelial Growth Factor Expression by Immunohistochemistry as a Possible Indicator of Prognosis in Invasive Breast Carcinoma of No Special Type.

Angiogenesis, the formation of new blood vessels from preexisting vascular network, is essential for tumor growth and spread. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor. To assess the expression of VEGF in invasive carcinoma of no special type and its correlation with all the known prognostic factors of breast carcinoma. Descriptive. Mastectomy specimens were studied noting the clinical details. The formalin-fixed tissues were subjected to routine processing and hematoxylin and eosin sections and studied extensively for all the histological prognostic factors. Representative sections from each case with the tumor were subjected to immunohistochemistry (IHC) staining with VEGF, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu) antibodies. Descriptive statistics, Chi-square tests, contingency table analysis using SPSS for Windows. One hundred and twelve cases of invasive carcinoma of special type were studied to evaluate various clinicopathological parameters. The association of VEGF with clinicopathological parameters and all the known prognostic factors was studied to note its significance. VEGF overexpression was observed in 69% of the cases. It was noted that larger tumor size, higher histological grade, lymphovascular invasion, nodal involvement, tumor necrosis, high microvessel density, ER negativity, PR negativity, and HER2/neu positivity had a significant statistical association with VEGF overexpression. We conclude that incorporating VEGF as a biomarker along with the known factors into a prognostic index will not only help predict clinical outcome more accurately, but also determines the patient who can be benefited with combinational therapy including anti-VEGF factors.

Abstract 18844: LncRNA MIR17HG and Peripheral Arterial Disease

Introduction: VEGF-A, a potent angiogenic growth factor has been extensively investigated as a therapeutic for Peripheral artery Disease (PAD). Recently, we have shown that increased levels of the anti-angiogenic VEGF-A isoform (VEGF165b) decreases perfusion recovery by inhibiting a truncated miR-17-20a cluster in ischemic endothelial cells and macrophages. Since miR-17-20a cluster is produced from the lncRNA-MIR17HG, we wanted to determine the role of VEGF165b-MIR17HG axis in regulating ischemic angiogenesis and perfusion recovery in PAD Methods: Hypoxia serum starvation in Human Umbilical vein endothelial cells (HUVECs) and Human microvascular endothelial cells (HMEC-1) was used as an in vitro PAD model. Results: Since LncRNAs have multiple transcript variants, we first determined if a specific MIR17HG transcript is present in human chronic limb threatening ischemia (CLTI) vs. normal. Bioinformatics analysis of a recently published bulk RNA-Seq data showed that among 17 annotated transcripts, transcript-1 (MIR17HG-Tr1) is specifically expressed in normal and CLTI muscle biopsies. Analysis of FPKMs showed a numerical increase in MIR17HG-Tr1 in CLTI vs. normal biopsies. qPCR analysis using primers specific for MIR17HG-Tr1 showed significantly higher expression of MIR17HG-Tr1 in HSS-HUVECs and HSS-HMEC-1 vs. their respective controls. VEGF165b inhibition in HSS-HUVECs and HSS-HMECs significantly decreased MIR17HG-Tr1 expression compared to IgG controls. To determine whether MIR17HG functions independent of its miRNA cluster, we silenced MIR17HG-Tr1 and examined miR17-92 cluster expression. Silencing MIR17HG-Tr1 didn’t significantly affect the expression of miR-17-92 cluster members either under normoxic or HSS conditions. In vitro tube formation assay showed that silencing MIR17HG-Tr1 significantly increased the angiogenic capacity of HSS-HUVECs vs. control. Conclusion: This data indicates VEGF165b inhibits MIR17HG-Tr1 to induce ischemic angiogenesis independently of its miR17-92 cluster. Ongoing studies are aimed at understanding the mechanism by which 1) VEGF165b regulates MIR17HG-Tr1 and 2) MIR17HG-Tr1 regulates revascularization in PAD.

Transdermal delivery of bFGF with sonophoresis facilitated by chitosan nanocarriers

The basic fibroblast growth factor (bFGF) is a potent angiogenic growth factor. Supplemental bFGF is essential for healing damaged skin, but its therapeutic use has been limited owing to its inherent instability. In this study, we propose a transdermal delivery system using sonophoresis along with the use of carboxymethyl chitosan nanocarriers which enables the administration of bFGF over a wide area of skin. The transdermal effect of the low-frequency ultrasound-enhanced delivery system was demonstrated in ex vivo experiments using hairless mouse skin. The subcutaneous administration of chitosan nanoparticles was evaluated by using fluorescence-labeled chitosan. To stably load bFGF into chitosan nanoparticles, a carrier with carboxymethyl chitosan was synthesized under a pH of around 7. Based on the results, we evaluated the effectiveness of transdermal drug delivery using sonophoresis. In skin cryosections, bFGF-loaded carboxymethyl chitosan nanocarriers delivered by ultrasound irradiation were present subcutaneously. We analyzed the bFGF content in skin tissue by ELISA and found that bFGF penetration in skin irradiated by ultrasound with nanocarriers. Penetration of bFGF-leaded carboxymethyl chitosan nanocarriers by ultrasound was significantly higher than that by ultrasound irradiation alone. Our results lay the groundwork for further utilization of polymeric nanocarriers using sonophoresis for the administration of biomacromolecules.

Downregulation of Xeroderma Pigmentosum Complementation Group C Expression by 17-Allylamino-17-Demethoxygeldanamycin Enhances Bevacizumab-Induced Cytotoxicity in Human Lung Cancer Cells

Introduction: Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor involved in nucleotide excision repair and regulation of non-small-cell lung cancer (NSCLC) cell proliferation and viability. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) blocks ATP binding to heat shock protein 90 (Hsp90), resulting in destabilization of Hsp90-client protein complexes. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor expressed by many types of tumors. Bevacizumab (Avastin) is a humanized monoclonal antibody against human VEGF used as an antiangiogenesis agent in the therapy of many cancers, proving successful in increasing objective tumor response rate and prolonging overall survival in NSCLC patients. Methods: After the bevacizumab and/or 17-AAG treatment, the expressions of XPC mRNA were determined by quantitative real-time PCR analysis. Protein levels of XPC and phospho-AKT were determined by Western blot analysis. We used specific XPC small interfering RNA and PI3K inhibitor (LY294002) to examine the role of the AKT-XPC signal in regulating the chemosensitivity of bevacizumab and 17-AAG. Cell viability was assessed by the MTS assay and trypan blue exclusion assay. Results: In this study, bevacizumab decreased XPC expression in human lung squamous cell carcinoma H520 and H1703 cells via AKT inactivation. Enhancement of AKT activity by transfection with constitutively active AKT vectors increased XPC expression and cell survival after treatment with bevacizumab. In addition, 17-AAG synergistically enhanced bevacizumab-induced cytotoxicity and cell growth inhibition in H520 and H1703 cells, associated with downregulation of XPC expression and inactivation of AKT. Discussion/Conclusion: Together, these results may provide a rationale to combine bevacizumab with Hsp90 inhibitors in future to enhance therapeutic effects for lung cancer.

Role of Angiopoietin-2 in Vascular Physiology and Pathophysiology.

Angiopoietins 1–4 (Ang1–4) represent an important family of growth factors, whose activities are mediated through the tyrosine kinase receptors, Tie1 and Tie2. The best characterized are angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 is a potent angiogenic growth factor signaling through Tie2, whereas Ang2 was initially identified as a vascular disruptive agent with antagonistic activity through the same receptor. Recent data demonstrates that Ang2 has context-dependent agonist activities. Ang2 plays important roles in physiological processes and the deregulation of its expression is characteristic of several diseases. In this review, we summarize the activity of Ang2 on blood and lymphatic endothelial cells, its significance in human physiology and disease, and provide a current view of the molecular signaling pathways regulated by Ang2 in endothelial cells.

Decreased levels of miR-28-5p and miR-361-3p and increased levels of insulin-like growth factor 1 mRNA in mononuclear cells from patients with hereditary hemorrhagic telangiectasia 1.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder inherited in an autosomal dominant manner. Patients with HHT can develop vascular dysplasias called telangiectasias and arteriovenous malformations (AVMs). Our objective was to profile and characterize micro-RNAs (miRNAs), short noncoding RNAs that regulate gene expression posttranscriptionally, in HHT patient-derived peripheral blood mononuclear cells (PBMCs). PBMCs, comprised mostly of lymphocytes and monocytes, have been reported to be dysfunctional in HHT. A total of 40 clinically confirmed HHT patients and 22 controls were enrolled in this study. PBMCs were isolated from 16 mL of peripheral blood and purified for total RNA. MiRNA expression profiling was conducted with a human miRNA array analysis. Select dysregulated miRNAs and miRNA targets were validated with reverse transcription-quantitative polymerase chain reaction. Of the 377 miRNAs screened, 41 dysregulated miRNAs were identified. Both miR-28-5p and miR-361-3p, known to target insulin-like growth factor 1 (IGF1), a potent angiogenic growth factor, were found to be significantly downregulated in HHT patients. Consequently, IGF1 mRNA levels were found to be significantly elevated. Our research successfully identified miRNA dysregulation and elevated IGF1 mRNA levels in PBMCs from HHT patients. This novel discovery represents a potential pathogenic mechanism that could be targeted to alleviate clinical manifestations of HHT.

Vascular Endothelial Growth Factor Isoform-B Stimulates Neurovascular Repair After Ischemic Stroke by Promoting the Function of Pericytes via Vascular Endothelial Growth Factor Receptor-1.

Ischemic stroke triggers endogenous angiogenic mechanisms, which correlates with longer survival in patients. As such, promoting angiogenesis appears to be a promising approach. Experimental studies investigated mostly the potent angiogenic factor vascular endothelial growth factor isoform-A (VEGF-A). However, VEGF-A increases the risk of destabilizing the brain microvasculature, thus hindering the translation of its usage in clinics. An attractive alternative VEGF isoform-B (VEGF-B) was recently reported to act as a survival factor rather than a potent angiogenic factor. In this study, we investigated the therapeutic potential of VEGF-B in ischemic stroke using different in vivo and in vitro approaches. We showed that the delayed intranasal administration of VEGF-B reduced neuronal damage and inflammation. Unexpectedly, VEGF-B stimulated the formation of stable brain microvasculature within the injured region by promoting the interaction between endothelial cells and pericytes. Our data indicate that the effects of VEGF-B were mediated via its specific receptor VEGF receptor-1 (VEGFR-1) that is predominately expressed in brain pericytes. Importantly, VEGF-B promoted the survival of pericytes, and not brain endothelial cells, by inducing expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and the main protein involved in energy homeostasis AMP-activated protein kinase α (AMPKα). Moreover, we showed that VEGF-B stimulated the pericytic release of factors stimulating a "reparative angiogenesis" that does not compromise microvasculature stability. Our study unraveled hitherto unknown role of VEGF-B/VEGFR-1 signaling in regulating the function of pericytes. Furthermore, our findings suggest that brain microvasculature stabilization via VEGF-B constitutes a safe therapeutic approach for ischemic stroke.

Type I collagen and its daughter peptides for targeting mucosal healing in ulcerative colitis: A new treatment strategy

Ulcerative colitis, particularly the chronic persistent form is characterized by the presence of active inflammation and extensive areas of ulceration in the colonic mucosa. The existing treatment protocol aims at only reducing intestinal inflammation, rather than targeting mucosal ulceration. In this study, type I collagen and its daughter peptides called collagen hydrolysate, highly popular reconstructive materials for tissue engineering applications, are hypothesized as healing matrices to target the recuperation of internal mucosal ulceration. The clinical assessments on day 10 of dextran sodium sulfate induced colitis in mice model revealed that both the collagen (1.56±0.29) and collagen hydrolysate treatments (1.33±0.33) showed a significant reduction in the rectal bleeding compared to the reference mesalamine treatment (2.50±0.33) and untreated negative control (2.40±0.40). VEGF, a potent angiogenic growth factor, over expressed during UC was down-regulated by collagen hydrolysate (1.06±0.25) and collagen (1.76±0.45) to a greater extent than by mesalamine (2.59±0.51) and untreated control (4.17±0.15). The down-regulation of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 also follows the same pattern. Histological observations were in accordance with the clinical indicators. Both collagen and collagen hydrolysate treatments showed significant reduction in mucosal damage score and facilitated faster regeneration of damaged mucosa.

Anti-angiogenic Therapy for Retinal Disease.

Recent breakthroughs in our understanding of the molecular pathophysiology of retinal vascular disease have allowed us to specifically target pathological angiogenesis while minimizing damage to the neurosensory retina. This is perhaps best exemplified by the development of therapies targeting the potent angiogenic growth factor and vascular permeability mediator, vascular endothelial growth factor (VEGF). Anti-VEGF therapies, initially introduced for the treatment of choroidal neovascularization in patients with age-related macular degeneration, have also had a dramatic impact on the management of retinal vascular disease and are currently an indispensable component for the treatment of macular edema in patients with diabetic eye disease and retinal vein occlusions. Emerging evidence supports expanding the use of therapies targeting VEGF for the treatment of retinal neovascularization in patients with diabetic retinopathy and retinopathy of prematurity. However, VEGF is among a growing list of angiogenic and vascular hyperpermeability factors that promote retinal vascular disease. Many of these mediators are expressed in response to stabilization of a single family of transcription factors, the hypoxia-inducible factors (HIFs), that regulate the expression of these angiogenic stimulators. Here we review the basic principles driving pathological angiogenesis and discuss the current state of retinal anti-angiogenic pharmacotherapy as well as future directions.

The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

In vitro evaluation of acellular porcine urinary bladder extracellular matrix – A potential scaffold in tissue engineered skin

Abstract Current tissue engineered skin substitutes employ a cellular scaffold, typically a collagen biopolymer to support the transfer of cells to the wound bed. These scaffolds however, exhibit poor approximation of epidermal–dermal histogenesis and delayed vascularisation upon implantation in vivo. In this study we evaluate a novel decellularised extracellular matrix (ECM) scaffold derived from the porcine urinary bladder wall; termed urinary bladder matrix (UBM). ECM scaffolds possess intrinsic angiogenic properties in the form of functional proteins and potent angiogenic growth factors. They have repeatedly demonstrated rapid angiogenesis and constructive remodelling in clinical trials. UBM is notable for containing an epithelial basement membrane, which offers improved approximation of epidermal–dermal histogenesis. In this study human dermal fibroblasts were seeded on UBM constructs in vitro. UBM ultrastructure was assessed using scanning electron microscopy and histology sections. Cell/scaffold interactions were examined, including cellular adhesion, proliferation, migration and protein synthesis using quantitative indicators. Cell morphology was determined using scanning electron microscopy. The study demonstrated that UBM supported the viability of human dermal fibroblasts. Cells displayed uninhibited proliferation and collagen synthesis was supported. Histological analysis showed the cells had organised spatially via the basement membrane. Cells grown on the basement membrane surface formed a monolayer while those grown on the opposite abluminal surface penetrated the scaffold. A high degree of cellular attachment was observed on the luminal and abluminal surfaces, 93.068 ± 2.3% and 92.045 ± 4.3%, respectively with no significant difference between the two surfaces ( P = 0.121, n = 3). This study demonstrates that UBM fulfils the fundamental properties for scaffold design and is a promising novel scaffold for future skin tissue engineering efforts.

Fluorescence‐based retention assays reveals sustained release of vascular endothelial growth factor from bone grafts

The sustained release of growth factors following their implantation in vivo is essential for successful outcomes in bone tissue engineering. In this study, we evaluated the release kinetics and delivery efficacies of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, incorporated into calcium phosphate bone grafts (BGs). We evaluated the release profile of VEGF from BGs using a novel fluorescence-based retention assay, which revealed that VEGF loaded on BGs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. In contrast, an ELISA-based release assay showed VEGF to have an early burst-release profile for the first week. However, the biological activity of VEGF released from the BGs was preserved over the 7-week release period, which is consistent with the sustained-release profile observed in the fluorescence-based retention assay. Furthermore, the in vivo bone-forming action of the VEGF-loaded BGs was well demonstrated in a rat subcutaneous model. Taken together, the sustained release of VEGF loaded onto BGs was effective in stimulating proliferation, angiogenesis and osteogenesis, suggesting the ultimate value of VEGF-engineered BGs for bone tissue engineering.

Abstract 491: Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The proto-oncogene MYCN is amplified in 25% of NB, and is associated with enhanced angiogenesis and poor survival. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor and, with the hepatocyte growth factor (HGF)/c-MET signalling pathway, is implicated in the progression of human NB. Cabozantinib (CBZ) is a small-molecule kinase inhibitor with potent activity against VEGFR2 and MET, currently in clinical trials against NB. In this study, the efficacy of CBZ was evaluated in tumors spontaneously arising in the TH-MYCN GEM model of high-risk NB, previously shown to faithfully recapitulate the radiological and hypervascular characteristics of childhood disease. TH-MYCN mice with spontaneous abdominal tumors were treated daily with either vehicle or 30mg/kg CBZ. Non-invasive T2-weighted anatomical MRI revealed that CBZ induced significant (p<0.001) tumor growth arrest after 48 hours compared to control. Quantitative functional MRI showed this volumetric response was associated with significant decreases in both the native longitudinal MRI relaxation time T1 (p<0.01) a measure of the ratio of bound to free water in tissue reflecting the increased extracellular space in tumors, and transverse MRI relaxation rate R2* (p<0.05), sensitive to paramagnetic deoxyhemoglobin. Histopathological analysis revealed a significant increase in necrosis (p<0.01), and significant decrease in microvessel density (p<0.01) and VEGFR2 scoring (p<0.02) in CBZ-treated tumors compared to control. Significant (p<0.01) tumor growth delay was maintained after 7 days treatment with CBZ compared to control, which translated into a significant improvement in survival rate (p<0.0001). This study demonstrates that CBZ is efficacious against, and conveys a survival benefit for the TH-MYCN GEM model of NB. The data reinforces native T1 as a biomarker of response to anti-vascular therapy due to its sensitivity to therapy-induced damage to erythrocytes and/or cell death-mediated increase in macromolecules into the extracellular space. Relatively fast R2* rates in NB arising in the TH-MYCN model are strongly associated with vascular instability, characterised by numerous regions of aggregated paramagnetic erythrocytes (“blood lakes”), and treatment-induced thrombosis with the pan-VEGFR inhibitor cediranib which increased tumor R2*. The CBZ-induced decrease in R2* is clearly associated with vascular disruption, hence precluding the extravasation of deoxygenated red blood cells. Citation Format: Gilberto S. Almeida, Philippa King, Yann Jamin, Albert Hallsworth, Hannah Webber, Sergey Popov, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2015-491

Abstract 1867: Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer

Abstract Combined hormone replacement therapy (HRT) containing progestins (P) and estrogens (E) has been shown to increase the risk of breast cancer in post-menopausal women (JNCI, 2000, 92:328). We showed that the commonly used P, medroxyprogesterone acetate (MPA), increases production of the potent angiogenic growth factor VEGF in breast cancer cells (Int J Cancer, 2001, 92:469). Furthermore, it has been established that P drives tumor progression in an in-vivo xenograft model (Can Res 2007; 67:9929). CD44 has recently been shown to be expressed by cancer stem cells (CSC) (Nature Reviews Cancer 2011; 11:254), and has therefore been used as a marker for detecting and enriching of tumor initiating cells. CD44 binds potent growth factors such as VEGF and acts as a co-receptor that helps mediate proliferative and migratory signaling for a variety of receptor tyrosine kinases. Thus, we hypothesized that MPA increases CD44 levels in breast cancer cells and promotes the emergence of self-renewing stem-cell-like-cells, which support MPA-accelerated tumors in vivo. Human T47-D human breast cancer cells were treated with MPA for 24h. Subsequent FACS analysis showed that MPA caused a significant increase in CD44 protein; CD44 was induced 10-fold (Control 5.9%, MPA 60.7%). A less robust, but significant increase also occurred in BT474 cells (Control 4.0%, MPA 11.2%). Further studies showed that MPA-induced CD44high cells also had high levels of ALDH enzyme activity, a characteristic of stem cells. CD44high cell induction was completely repressed by the anti-progestin RU-486, indicating involvement of progesterone receptors. Induction of CD44 protein occurred in a time-dependent manner, with a significant increase 6h after MPA treatment and reaching a peak after 24h. Interestingly, CD44high expression remained elevated for at least another 24 hours, even though MPA was removed from the treatment media, indicating that once induced, CD44 may be a stable protein. Importantly, various natural and synthetic P that are used widely in HRT prescriptions (Progesterone, Norethindrone, Norgestrel), brought about a similar increase in CD44high cells. Considering P has previously been linked to increased VEGF secretion by hormone-responsive cancer cells, we tested whether VEGF signaling plays a role in the elevation of CD44high cells. However, this process was found to be VEGF-independent. Our results show that natural and synthetic progestins induce a CD44high cell population which is likely associated with development of MPA-induced tumors. Thus targeted therapies against CD44 may be useful in controlling progression of P-driven tumors. Supported in part by COR award from the College of Veterinary Medicine, University of Missouri. Citation Format: Sandy Goyette, Benford Mafuvadze, Matthew T. Cook, Yayun Liang, Salman M. Hyder. Enrichment of CD44high stem-cell-like cells as a possible mechanism of progestin-dependent progression of human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1867. doi:10.1158/1538-7445.AM2015-1867

Multiple roles of hypoxia in ovarian function: roles of hypoxia-inducible factor-related and -unrelated signals during the luteal phase.

There is increasing interest in the role of oxygen conditions in the microenvironment of organs because of the discovery of a hypoxia-specific transcription factor, namely hypoxia-inducible factor (HIF) 1. Ovarian function has several phases that change day by day, including ovulation, follicular growth and corpus luteum formation and regression. These phases are regulated by many factors, including pituitary hormones and local hormones, such as steroids, peptides and cytokines, as well as oxygen conditions. Hypoxia strongly induces angiogenesis because transcription of the potent angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF1. Follicular development and luteal formation are accompanied by a marked increase in angiogenesis assisted by HIF1-VEGF signalling. Hypoxia is also one of the factors that induces luteolysis by suppressing progesterone synthesis and by promoting apoptosis of luteal cells. The present review focuses on recent studies of hypoxic conditions, as well as HIF1-regulated genes and proteins, in the regulation of ovarian function.

Chitosan–hyaluronic acid/VEGF loaded fibrin nanoparticles composite sponges for enhancing angiogenesis in wounds

Reduced levels of endogenous growth factors and diminished angiogenesis are contributory factors for impaired wound healing in diabetic patients. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor which stimulates multiple phases of wound healing angiogenesis and thereby accelerates healing. The aim of this work was to develop chitosan–hyaluronic acid composite sponge incorporated with fibrin nanoparticles loaded with VEGF as a wound dressing for diabetic wounds. VEGF loaded fibrin nanoparticles (150–180nm) were prepared and characterized which were then incorporated to the composite sponge. The prepared sponges were characterized by SEM and FT-IR. Porosity, swelling, biodegradation, mechanical properties and haemostatic potential of the sponges were also studied. Release of VEGF from the composite sponges was evaluated using ELISA kit. More than 60% of the loaded VEGF was released in three days. Cell viability and attachment studies of the composite sponges were evaluated using human dermal fibroblast (HDF) cells and human umbilical vein endothelial cells (HUVECs). HUVECs seeded on VEGF containing sponges showed capillary like tube formation which was absent in control sponges. The results suggest that the prepared chitosan–hyaluronic acid/VEGF loaded nanofibrin composite sponges (CHVFS) have potential to induce angiogenesis in wound healing.

Putative role of ischemic postconditioning in a rat model of limb ischemia and reperfusion: involvement of hypoxia-inducible factor-1α expression.

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It improves angiogenesis and tissue perfusion in ischemic skeletal muscle. In the present study, we tested the hypothesis that ischemic postconditioning is effective for salvaging ischemic skeletal muscle resulting from limb ischemia-reperfusion injury, and that the mechanism involves expression of HIF-1α. Wistar rats were randomly divided into three groups (n=36 each): sham-operated (group S), hindlimb ischemia-reperfusion (group IR), and ischemic postconditioning (group IPO). Each group was divided into subgroups (n=6) according to reperfusion time: immediate (0 h, T0), 1 h (T1), 3 h (T3), 6 h (T6), 12 h (T12), and 24 h (T24). In the IPO group, three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were carried out before reperfusion. At all reperfusion times (T0-T24), serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, as well as interleukin (IL)-6, IL-10, and tumor necrosis factor-α (TNF-α) concentrations, were measured in rats after they were killed. Histological and immunohistochemical methods were used to assess the skeletal muscle damage and HIF-1α expression in skeletal muscle ischemia. In groups IR and IPO, serum LDH and CK activities and TNF-α, IL-6, and IL-10 concentrations were all significantly increased compared to group S, and HIF-1α expression was up-regulated (P<0.05 or P<0.01). In group IPO, serum LDH and CK activities and TNF-α and IL-6 concentrations were significantly decreased, IL-10 concentration was increased, HlF-1α expression was down-regulated (P<0.05 or P<0.01), and the pathological changes were reduced compared to group IR. The present study suggests that ischemic postconditioning can reduce skeletal muscle damage caused by limb ischemia-reperfusion and that its mechanisms may be related to the involvement of HlF-1α in the limb ischemia-reperfusion injury-triggered inflammatory response.

VEGF expression, microvessel density and dendritic cell decrease in thyroid cancer

Thyroid cancer is one of the five most common cancers in the age between 20 and 50 years. Many factors including the potent angiogenic vascular endothelial growth factor (VEGF) and different dendritic cell types are known to be related to thyroid tumourogenesis. The study was performed to address the expression of VEGF and microvessel density in thyroid cancers and to evaluate the effect of VEGF expression in thyroid tumour cells on the dendritic cells. We investigated 65 patients with different types of thyroid carcinomas: papillary (PTC), oncocytic (OTC), follicular (FTC) and anaplastic (ATC), immunohistochemically with antibodies against VEGF, CD1a, CD83, S100 and CD31. Our results suggest that the expression of VEGF is significantly more often in PTC than ATC (92.3% vs. 60.0%, p = 0.025). The microvessel density marked with CD31 in the tumour border of PTC was significantly higher as compared to FTC (p = 0.039), but not to ATC and OTC (p = 0.337 and 0.134). We found that CD1a- and CD83-positive cells were dispersed with variable density and in OC CD31+ vessel numbers were positively correlated with CD83+ dendritic cells in tumour stroma (R = 0.847, p = 0.016). We did not find statistically significant associations of the survival of patients with PTC after the surgical therapy with VEGF expression and MVD. In conclusion we may state that VEGF expression in tumour cells of thyroid cancer can induce neovascularization and suppress dendritic cells.

VEGF and Colon Cancer Growth Beyond Angiogenesis: Does VEGF Directly Mediate Colon Cancer Growth Via a Non-angiogenic Mechanism?

In this article we review the role of vascular endothelial growth factor (VEGF) in colon cancer growth and the underlying mechanisms. Angiogenesis, the growth of new capillary blood vessels in the body, is critical for tissue injury healing and cancer growth. In 1971, Judah Folkman proposed the concept that tumor growth beyond 2 mm is critically dependent on angiogenesis. Tumors including colon cancers release angiogenic growth factors that stimulate blood vessels to grow into the tumors thus providing oxygen and nutrients that enable exponential growth. VEGF is the most potent angiogenic growth factor. Several studies have highlighted the role of VEGF in colon cancer, specifically in the stimulation of angiogenesis. This role of VEGF is strongly supported by studies showing that inhibition of VEGF using the blocking antibody, bevacizumab, results in decreased angiogenesis and abrogation of cancer growth. In the United States, bevacizumab in combination with chemotherapy is FDA approved for the treatment of metastatic colon cancer. However, the source of VEGF in colon cancer tissue, the mechanisms of VEGF generation in colon cancer cells and the molecular pathways involved in VEGF mediated angiogenesis in colon cancer are not fully known. The possibility that VEGF directly stimulates cancer cell growth in an autocrine manner has not been explored in depth.

Endothelial progenitor cells in clinical settings.

Senescence of cells is associated with shortened or damaged telomeres and is characterized by permanent exit from the cell cycle and altered function. Cellular senescence is caused by repeated cell division, and also conditions of stress including inflammation and reactive oxygen species can lead to the development of premature senescence. At the cellular level, proliferative and oxidative-stress induced cell senescence related to a pro-inflammatory state might strongly contribute to age-associated impaired tissue and organ functions. Vascular cells (endothelial cells, vascular smooth muscle cells) and bone marrow-derived endothelial progenitor cells have been repeatedly shown to have pivotal role in the maintenance and regeneration of cardiovascular tissue. Therefore, the molecular mechanisms of vascular cell senescence have been extensively studied. However, therapeutic approaches to prevent cellular senescence in cardiovascular disease (CVD) are still limited. Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) are all potent angiogenic growth factors in animal models of ischemia, but their therapeutic effects are not the same in animal experiments and clinical trials. A multicenter, double-blind, placebo-controlled phase III clinical trial in Japan and a US phase II clinical trial of HGF gene therapy for critical limb ischemia (CLI) demonstrated a significant improvement in primary end points and an increase in transcutaneous partial pressure of oxygen even after one year compared with placebo, whereas effectiveness of VEGF and FGF treatment for CLI has not yet been shown. Moreover, our recent publication and another researcher demonstrated that HGF acts as an anti-inflammatory cytokine, while VEGF and FGF act as pro-inflammatory cytokine. This review overviews the outcomes of clinical trials using angiogenic growth factors, which have shown a dramatic effect in several animal studies. Additionally, interventions with HGF aimed at improving the regenerative capacity of stem/progenitor cells and vascular cells by preventing cellular senescence are discussed.