VEGFR2 Gene Research Articles

Vascular anomalies

Vascular anomalies

Tyrosine kinases as possible therapeutic targets in pulmonary artery intimal sarcoma

10055 Background: Pulmonary artery intimal sarcomas (PAIS) are very rare, highly malignant and clinically aggressive tumors, with yet elusive underlying pathogenic mechanisms and histogenesis. Our aim was to gain molecular insight in the oncogenic events leading to PAIS development, and to investigate the possibility of a pharmacological approach for the treatment of PAIS, specifically targeting the receptor tyrosine kinases (RTK) activated in these tumors. Methods: Fourteen cases of advanced high grade poorly differentiated PAIS were analyzed by immunohistochemistry and selectively by a variety of cytogenetic/molecular techniques, e.g. karyotyping, FISH, mRNA in situ hybridization, array-CGH, tyrosine kinases RT-PCR assay and Western blotting analysis. The study was supplemented by sequencing of PDGFRA, PDGFRB, KIT, VEGFR-1, VEGFR-2 and EGFR genes. Ex vivo functional assays were applied to test the sensitivity of PAIS primary tumor cells to different, clinically approved RTK inhibitors. Results: We demonstrate that (1) PAIS do not have mutations within the kinase domains of PDGFRs, KIT, VEGFRs or EGFR, (2) all cases show a similar cytogenetic molecular profile, characterized by amplification of PDGFRA/KIT/VEGFR2 and MDM2/CDK4 genes, while EGFR, PDGFRB and VEGFR1 are frequently polysomic but rarely highly amplified, (3) high level of expression of PDGFRA, VEGFR1/VEGFR2 and EGFR is uniformly present in the tested tumors, (4) PAIS demonstrate activation (phosphorylation) of PDGFRA and downstream PI3K-AKT and MAP-Kinase signaling pathways, (4) in ex vivo assays, a potent effect of BMS354825 on proliferation and survival of primary cells of PAIS is proven. Conclusions: PDGFRA is amplified and activated in PAIS, thus constituting one of many possible targets for the therapy. Tested inhibitors showed differential efficacy for inhibition of PAIS cells by ex vivo assays. No significant financial relationships to disclose.

Hypoxia-inducible Factors in the First Trimester Human Lung

Lung development takes place in a relatively low-oxygen environment, which is beneficial for lung organogenesis, including vascular development. Hypoxia-inducible factor (HIF)-1 plays an important role in mediating oxygen-regulated events. HIF-1 is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau (VHL) tumor suppressor protein leads to rapid degradation of the HIF-1alpha subunit. Interaction with VHL requires hydroxylation of HIF-1alpha proline residues by prolyl hydroxylases (PHDs). We investigated the expression of the various components regulating HIF-1alpha stability in first trimester (8-14 weeks) human lungs. Spatial expression was assessed by immunohistochemistry and temporal expression by quantitative PCR. Immunoreactivity for PHD1, PHD3, and seven in absentia homolog (SIAH)1 was noted in the pulmonary epithelium. PHD2 was not expressed in the airway epithelium, but in the lung parenchyma. HIF-1alpha and vascular endothelial growth factor (VEGF) immunoreactivity were primarily detected in the branching epithelium. HIF-2alpha and ARNT proteins localized to the developing epithelium as well as mesenchymal, most likely vascular, structures in the parenchyma. VEGF receptor 2 (VEGFR2) was found in the subepithelium as well as in vascular structures of the mesenchyme. All components of the VEC complex (VHL, NEDD8, and Cullin2) were found in the epithelium. Quantitative PCR analysis demonstrated that VEGF, VEGFR1, HIF-1alpha, HIF-2alpha, ARNT, PHD1, PHD2, PHD3, and SIAH1 gene expression was constant during early pulmonary organogenesis. Cumulatively, the data suggest that the lung develops in a low-oxygen environment that allows for proper vascular development through HIF-regulated pathways.

Stem cell-related cardiac gene expression early after murine myocardial infarction

Clinical experimental stem cell therapy after myocardial infarction appears feasible, but its use has preceded the understanding of the working mechanism. The ischemic recipient cardiac environment is determinative for the attraction and subsequent fate of stem cells. Here, we studied expression levels of genes that are anticipated to be essential for adequate stem cell-based cardiac repair at various time-points during the 1 month period following myocardial infarction (MI). Gene expression in the hearts of mice that underwent MI by permanent or transient (30 min) ligation of the coronary artery was monitored using quantitative RT-PCR analysis of mRNA isolated from whole heart sections as well as from specific, laser micro-dissected, regions of sections. Protein expression was performed by immunohistochemical stainings and Western blot analysis. Many inflammatory genes were highly expressed for at least 1 week after MI. The expression of pro-angiogenic genes such as bFGF, VEGF-A and VEGF-R2 changed only marginally post-MI. Markers used to test stem cell gene expression remained unchanged post-MI with the exception of G-CSF and GM-CSF, which are genes that are also known to enhance the inflammatory response. Analysis of micro-dissected regions revealed that SDF-1, SCF (both stem cell attractants) and VEGF-R2 (involved in angiogenesis) gene expression was slightly decreased especially in the infarcted region. Genes that are generally considered to participate in stem cell-related processes and angiogenesis were not upregulated after MI, whereas the inflammatory gene expression dominated. Modulation of this imbalance might be of value for stem cell-mediated therapy.

VEGFR1 (Flt-1+/−) gene knockout leads to the disruption of VEGF-mediated signaling through the nitric oxide/heme oxygenase pathway in ischemic preconditioned myocardium

This report demonstrates that mice deficient in Flt-1 failed to establish ischemic preconditioning (PC)-mediated cardioprotection in isolated working buffer-perfused ischemic/reperfused (I/R) hearts compared to wild type (WT) subjected to the same PC protocol. WT and Flt-1+/− mice were divided into four groups: (1) WT I/R, (2) WT + PC, (3) Flt-1+/− I/R, and (4) Flt-1+/− + PC. Group 1 and 3 mice were subjected to 30 min of ischemia followed by 2 h of reperfusion and group 2 and 4 mice were subjected to four episodes of 4-min global ischemia followed by 6 min of reperfusion before ischemia/reperfusion. For both wild-type and Flt-1+/− mice, the postischemic functional recovery for the hearts was lower than the baseline, but the recovery for the knockout mice was less compared to the WT mice even in preconditioning. The myocardial infarction and apoptosis were higher in Flt-1+/− compared to wild-type I/R. Flt-1+/− KO mice demonstrated pronounced inhibition of the expression of iNOS, p-AKT & p-eNOS. Significant inhibition of STAT3 & CREB were also observed along with the inhibition of HO-1 mRNA. Results demonstrate that Flt-1+/− mouse hearts are more susceptible to ischemia/reperfusion injury and also document that preconditioning is not as effective as found in WT and therefore suggest the importance of VEGF/Flt-1 signaling in ischemic/reperfused myocardium.

Anti-fibrotic effects of tetrandrine on bile-duct ligated rats

Tetrandrine (Tet) (C38H42O8N2; molecular weight, 622), an alkaloid isolated from the Chinese medicinal herb Stephania tetrandra, has been shown to elicit anti-inflammatory and anti-fibrotic effects in pulmonary diseases, but the mechanism of action has yet to be investigated. In this study, we tested whether Tet exerts anti-fibrotic effects on rat hepatic fibrosis through anti-NFkappaB pathways. After bile-duct ligation, rats were given Tet (1 or 5 mg/kg) or silymarin (50 mg/kg, as a positive control) by gavage twice daily for 3 weeks. Liver sections were taken for Sirius red quantitative scoring, immunofluorescence double staining of alpha-smooth muscle actin (alpha-SMA) and NFkappaB, and for quantitative determinations of the mRNA expression levels of TGF-beta1, alpha-SMA, collagen 1alpha2, inducible nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), metallothionein, vascular endothelial growth factor (VEGF), and VEGF type II receptor (VEGFR2) genes. The results showed that both Tet and silymarin treatment significantly reduced the fibrosis scores and hepatic collagen content of BDL rats, compared with no treatment. Both Tet and silymarin treatments decreased the number of alpha-SMA- and NFkappaB-positive cells in fibrotic livers. Moreover, Tet and silymarin treatments attenuated the mRNA expression levels of TGF-beta1,alpha-SMA, collagen 1alpha2, iNOS, ICAM-1, VEGF, and VEGFR2 genes, and induced the mRNA expression of the metallothionein gene. This study suggests that the anti-fibrotic effects of Tet were related to the reduction of fibrosis-related gene transcription, the attenuation of NFkappaB-activated pathways, and the induction of metallothionein gene transcription in the livers of BDL rats.

Of mice and men; the translational physiology of a genetic form of lymphoedema

Of mice and men; the translational physiology of a genetic form of lymphoedema

Chrysin Inhibits Lipopolysaccharide-Induced Angiogenesis via Down-Regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R Pathways

The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The IN VIVO CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10(-8) - 10(-5) M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both IN VITRO and IN VIVO LPS-induced angiogenesis.

Association between genetic variations of vascular endothelial growth factor receptor 2 and atopy in the Korean population

Vascular endothelial growth factor (VEGF) has been suggested to be a key mediator in the development of atopy and T(H)2 inflammation. We sought to evaluate the effects of variations in the gene coding VEGF receptor (VEGFR) 2 on intermediate phenotypes of asthma in the Korean population. A cohort of 2055 children and adolescents responded to a questionnaire concerning asthma symptoms and risk factors and underwent methacholine bronchial challenge and skin tests. The VEGFR2 gene, including the promoter area, was sequenced on 24 healthy subjects to discover informative single nucleotide polymorphisms (SNPs; minor allele frequency >2%). After haplotype reconstruction, 4 tagging SNPs (IVS6+54A>G, +889G>A, +1416T>A, and IVS25-92G>A) were scored. These SNPs were also scored in 480 adult asthmatic patients to verify the above genetic association study. The prevalence of atopy was associated with a single SNP (+889G>A) of VEGFR2 with borderline significance (P = .048; relative risk, 1.13; 95% CI, 1.00-1.28). However, haplotype analysis showed that the atopy prevalence was strongly associated with a haplotype (AGAG) of VEGFR2 (P = .002; relative risk, 1.25; 95% CI, 1.09-1.42). As for airway hyperresponsiveness, neither individual SNPs nor haplotypes were found to be associated. Interestingly, the significant association was also found between atopy and the AGAG haplotype among adult asthmatic patients (P = .008; odds ratio, 1.66; 95% CI, 1.14-2.44). The present study demonstrated that genetic variations of VEGFR2 are significantly associated with atopy in the Korean population.

Hypoxia Inducible Factor-1α Is over Expressed in CLL B Cells Because of an Impaired Proteasome Pathway Associated with Defective Interaction with von Hippel-Landau Protein.

We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

Silibinin Inhibits Angiogenesis via Flt-1, but not KDR, Receptor Up-Regulation 1

Our previous study found that silymarin (SM) and its major pure component silibinin (SB) have anti-angiogenic effects via decreased vascular endothelium growth factor (VEGF) secretion of LoVo cells (colon cancer). We designed this consecutive study to evaluate the anti-angiogenic effects of SM/SB in vivo, and on VEGF receptor (VEGFR) gene expression. We used LoVo cells exposed to SM/SB in a modified chicken chorioallantoic membrane assay (CAM) to evaluate anti-angiogenic effects. We used EA.hy 926 cells (endothelial cells) exposed to SM/SB to evaluate the effect on VEGFR-1 (Flt-1) and VEGFR-2 (KDR), with 1-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). In CAM, SM/SB showed a dose-dependent decrease on the vascular density index (VDI) induced by LoVo cells, as did thalidomide in a concentration of 10 microg/ml. Adding escalating dosages of VEGF successfully reversed this inhibitory effect. RT-PCR revealed that SB up-regulated Flt-1 mRNA expression of EA.hy 926 cells. SM had a similar trend, although the effect was not statistically significant (P = 0.19). Neither drug effected KDR mRNA expression. We conclude that anti-angiogenic effects of SM/SB are associated with the up-regulation of VEGFR-1 (Flt-1) gene expression and that they are good candidates for combination therapy to treat colorectal cancer.

Gene expression profile of cytokine and growth factor during differentiation of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells (MSCs), which are adherent stromal cells of a nonhematopoietic origin, have the ability to give rise to various differentiated cell types. MSCs regulate localization, self-renewal and differentiation of hematopoietic stem cells (HSCs) due to MSCs' secretion of cytokines and growth factors, the cell-to-cell interactions and the influence of the extracellular matrix proteins. Using RT-PCR analysis, we examined the expression levels of cytokines and growth factors from MSCs and their differentiated cell types, including osteoblasts, adipocytes and endothelial cells. Cytokine and growth factor genes, including IL-6, IL-8, IL-11, IL-12, IL-14, IL-15, LIF, G-CSF, GM-CSF, M-SCF, FL and SCF, were found to be expressed in the MSCs. In contrast, there was no IL-1α, IL-1β, or IL-7 expression observed. The IL-12, IL-14, G-CSF, and GM-CSF mRNA expression levels either disappeared or decreased after the MSCs differentiated into osteoblasts, adipocytes, and endothelial cells. Among the differentiated cells derived from MSCs, osteoblasts, adipocytes, and endothelial cells expressed the osteopontin, aP 2, and the VEGFR-2 gene, respectively. These profiles could help determine future clinical applications of MSCs and their derivatives for cell therapy.

Temporal renal expression of angiogenic growth factors and their receptors in experimental diabetes

It has been postulated that vascular endothelial growth factor (VEGF) plays a role in the progression of renal injury. However, the role of other angiogenic factors and their receptors, such as the angiopoietins and Tie2, and in particular their relation to renoprotective therapies, such as agents that interrupt the renin-angiotensin system, have not been studied in the context of diabetes-related renal injury. Renal expression of VEGF, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors, VEGF-R2 and Tie-2, were assessed using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting, in control and streptozotocin diabetic rats, untreated or receiving the AT1 receptor antagonist, valsartan, or the AT2 receptor antagonist, PD123319. Diabetes was associated with increased gene and protein expression of VEGF, VEGF-R2, Ang-1, Ang-2 and Tie-2. AT1 receptor antagonism attenuated gene expression of these cytokines and receptors, yet PD123319, which had no effect on blood pressure, reduced VEGF-R2 and Ang-1 gene expression and decreased VEGF, Ang-1 and Ang-2 protein levels. In experimental diabetes, there is significant upregulation within the kidney of various angiogenic cytokines and their receptors. Furthermore, the effects of angiotensin II receptor blockade on these parameters is consistent with the VEGF-VEGF-R2 and angiopoietin-Tie-2 axes being modulated in the kidney by haemodynamic factors in the diabetic context.

Blood Outgrowth Endothelial Cells (BOEC) Contribute to Tumor Vascularization and Can Be Used for Delivery of Cancer Gene Therapy.

Continuous and adequate levels of angiogenesis inhibitors at the site of tumor vasculature may be necessary to obtain optimal antitumor effect. Following recent observations that circulating in blood endothelial cells enhance tumor vasculogenesis we have designed grown from peripheral blood endothelial cells to deliver anti-angiogenic antitumor therapy. BOEC, after several-fold expansion, expressed endothelial markers: VE-cadherin, CD31, von Willebrand factor, and P1H12 and responded to functional endothelial assays: uptake of acetylated low-density lipoprotein, and vascular tube formation. BOEC were also found in functional vessels in vivo, when NOD-SCID mice implanted subcutaneously with Lewis lung carcinoma or H226 lung cancer, were injected via tail vein with 5x105 BOEC. Staining for CD31 on tumor sections of BOEC injected mice demonstrated increased blood vessel density (BOEC 1982±1281, sham 328±229 (P<0.001)). In FsaII tumor model, systemic BOEC injection resulted in increased tumor volumes and higher tumor oxygenation when compared with sham injected animals. Injected via tail vein BOEC distributed initially throughout organs with high vascularization, but in 72 hours remained only in tumor, liver, spleen, and bone marrow. Given the ability of BOEC to survive within tumor vasculature, we designed BOEC to deliver endostatin and soluble VEGFR-1 (sFlt1) genes to the tumor's endothelium. A retroviral MSCV vector was constructed to contain green fluorescent protein (GFP) and human endostatin gene (hES) or sFlt1. The hES or sFlt1 containing BOEC were evaluated for vWF, CD31, and VE-cadherin, and found positive. Transduced with endostatin or sFlt1 gene BOEC had a slower growth rate in culture, produced detectable endostatin (1.84 ng/ml) or sFlt-1 (665 pg/ml), and were able to inhibit MVEC growth in co-culture condition. When endostatin transduced BOEC were injected to mice with tumor tumor vascularization was decreased (EBOEC 290±172 versus BOEC 1982±1281(p<0.001)). This resulted in tumor volume reduction in mice injected with EBOEC (0.364±0.227cm3 versus sham 0.802±0.431cm3 (p=0.102)). This early data suggest that BOEC have the potential for tumor-specific delivery of cancer gene therapy.

Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer.

Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects. The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with N(G)-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10(7) PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A. Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.

Anti-angiogenesis effect on glioma of attenuated Salmonella typhimurium vaccine strain with flk-1 gene.

To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.

Screening and confirmation of differentially expressed genes in placentas from PIH patients

目的 了解妊娠高血压综合征(妊高征)胎盘组织基因表达的改变,确定与妊高征发病相关的基因,为研究妊高征的发病机理提供线索. 方法收集2001年3月至2002年9月西安地区以剖宫产终止妊娠的42例妊高征患者及22例正常产妇的产前、产后临床资料;收集胎盘组织,制作胎盘组织芯片;建立妊高征患者数据库.采用抑制消减杂交技术(suppression subtractive hybridi-zation, SSH)建立妊高征胎盘组织cDNA消减文库;利用差异筛选技术筛选出妊高征胎盘高表达基因,用RT-PCR和免疫组织化学技术验证部分高表达基因. 结果建立了由42例中、重度妊高征患者和22例正常产妇组成的数据库; 用66例胎盘组织制成150点的胎盘组织芯片.利用SSH和差异筛选共分离出103个阳性克隆,测序成功90个.测序结果显示90个cDNA片断与GenBank/EMBL中36条已知功能基因有95%以上的同源性. 结论妊高征患者胎盘组织基因表达发生较大变化;有必要研究血管内皮生长因子受体、卵巢抑制素、丝氨酸蛋白酶抑制素、细胞角蛋白7以了解妊高征的发病机理。

Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets.

Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To survive and function, transplanted islets must revascularize because islet isolation severs arterial and venous connections; the current paradigm is that islet revascularization originates from the transplant recipient. Because isolated islets retain intraislet endothelial cells, we determined whether these endothelial cells contribute to the revascularization using a murine model with tagged endothelial cells (lacZ knock-in to Flk-1/VEGFR2 gene) and using transplanted human islets. At 3-5 weeks after transplantation beneath the renal capsule, we found that islets were revascularized and that the transplant recipient vasculature indeed contributed to the revascularization process. Using the lacZ-tagged endothelial cell model, we found that intraislet endothelial cells not only survived after transplantation but became a functional part of revascularized islet graft. A similar contribution of intraislet endothelial cells was also seen with human islets transplanted into an immunodeficient mouse model. In the murine model, individual blood vessels within the islet graft consisted of donor or recipient endothelial cells or were a chimera of donor and recipient endothelial cells, indicating that both sources of endothelial cells contribute to the new vasculature. These observations suggest that interventions to activate, amplify, or sustain intraislet endothelial cells before and after transplantation may facilitate islet revascularization, enhance islet survival, and improve islet transplantation.

Crude Extract of Salvia miltiorrhiza and Salvianolic Acid B Enhance in Vitro Angiogenesis in Murine SVR Endothelial Cell Line

Salvia miltiorrhiza (SM) has been used clinically in Asian countries to improve the microcirculation in the human body. Although a pure compound extracted from SM, salvianolic acid B (Sal B), has been reported to be effective against fibrosis and ischemia-reperfusion injury, possibly through its anti-lipid peroxidation action, the effect of SM on angiogenesis remains unclear. It is our interest to investigate the role of SM on the regulation of the angiogenic process. By using the SVR endothelial cell line as an in vitro system, the effects of Sal B on the gene expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, VEGF-R2 were evaluated by morphology, differentiation assay, reverse-transcribed polymerase chain reaction (RT-PCR) and Western blot analysis. The results showed that both the crude extract of SM and the pure compound Sal B had enhancing effects on cell growth and differentiation. The gene expression of matrix metalloproteinase-2 (MMP-2) was up-regulated after Sal B treatment for 2 h, while VEGF and VEGF-R2 gene expression were up-regulated 40 min after Sal B treatment. We conclude that the crude extract of SM and Sal B enhance angiogenic processes on SVR cells through up-regulation of VEGF and VEGF receptors genes.

3P-0621 Vascular cell senescence induces vascular inflammation in human atherosclerosis

Neuronal and vascular cells share common chemical signals. Vascular endothelial growth factor (VEGF)-C and -D and their receptor VEGFR-3/Flt-4 mediate lymphangiogenesis, but they occur also in the brain. Quantitative RT-PCR of mouse brain tissues and cultivated cells showed that the VEGFR-3 gene is highest transcribed in postnatal brain and in glial precursor cells whereas VEGF-C and -D are variably produced by different neuronal and glial cells. In neurospheres (neural stem cells) VEGFR-3 was induced by differentiation with platelet-derived growth factor (PDGF). In functional studies with an A2B5- and nestin-positive, O4-negative murine glial precursor cell line, VEGF-C and -D stimulated phosphorylation of the kinases Erk1/2; this signal transduction was inhibited by UO126. Both peptides induced the proliferation of glial precursor cells which could be inhibited by UO126. Furthermore, VEGF-D considerably enhanced their migration into an open space in a wound-healing assay. These results show that VEGF-C/-D together with its receptor VEGFR-3 provides an auto-/paracrine growth and chemotactic system for glial precursors in the developing brain.