Histophysiological effects of fluid resuscitation on heart, lung and brain tissues in rats with hypovolemia

The efficacy of using colloids and crystalloids in the treatment of hypovolemia still remains controversial. An important aspect in treating hypovolemia is to re-establish normal tissue hemodynamics after fluid resuscitation. Production of nitric oxide (NO) or growth factors such as transforming growth factor beta (TGF-beta) has been identified as a key mechanism in physiological and pathological processes in the different systems. This study was designed to investigate the histophysiological effects of resuscitation with different plasma substitutes on the heart, lung and brain tissues following acute blood loss in male Sprague-Dawley rats weighing 250-280g (n=30). After anesthesia with sodium pentobarbital, the left femoral vein and artery were cannulated for the administration of volume expanders and for direct measurement of arterial pressure and heart rate. Twenty rats were bled (5ml/10min) and infused (5ml/10min) with one of four randomly selected solutions, (a) human albumin, (b) gelatin (Gelofusine), (c) dextran-70 (Macrodex); or (d) physiological saline (0.9% isotonic saline). Five control rats were bled without infusion. Tissue samples were taken and fixed in 10% formalin solution, then processed for embedding in paraffin wax. Sections were cut and stained with hematoxylin and eosin. Indirect immunohistochemical labelling was performed to reveal binding of primary antibodies against endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and TGF-beta. Mild immunoreactivity of eNOS was observed in endothelial cells of vessels in brain, heart and lung tissues. Increased immunoreactivities of eNOS, iNOS and TGF-beta were observed in the non-fluid resuscitated group in these organs; mild, moderate, moderate and strong immunoreactivities were seen in the albumin, gelatin, physiological saline and dextran-70 treated groups, respectively. Immunoreactivities of iNOS and TGF-beta in the non-fluid resuscitated group were increased significantly, in comparison to the other groups, apart from the dextran-70 treated group. The results of this study show that gelatin solution and physiological saline may be of use after acute blood loss, and dextran-70 is not the preferred resuscitation fluid in the early stages of acute blood loss. It was concluded that albumin solution is the preferred fluid for resuscitation.     

胎盘生长因子(PLGF)及其受体血管内皮生长因子受体-1(VEGFR-1)在子宫内膜异位症中的表达及意义

目的探讨胎盘生长因子(PLGF)及其受体血管内皮生长因子受体-1(VEGFR-1)与子宫内膜异位症(endom etriosis,EMS)发病的相关性。方法用免疫组织化学染色法检测28例子宫内膜异位症患者的在位子宫内膜和异位子宫内膜(增生期、分泌期各14例),以及28例正常子宫内膜(增生期15例,分泌期13例)中PLGF及其受体VEGFR-1的表达。结果PLGF、VEGFR-1在各组内膜中均有表达,分泌期表达高于增生期,有显著差异。EMS的异位子宫内膜中的表达显著高于其在位子宫内膜和正常子宫内膜;EMS的在位子宫内膜和正常子宫内膜之间比较无明显差异。结论PLGF、VEGFR-1可能参与子宫内膜异位症的发病过程。     

Vascular Endothelial Growth Factor Receptor-1 Modulates Vascular Endothelial Growth Factor-Mediated Angiogenesis via Nitric Oxide

The known responses of vascular endothelial growth factor (VEGF) are mediated through VEGF receptor-2 (VEGFR-2/KDR) in endothelial cells. However, it is unknown whether VEGFR-1 (Flt-1) is an inert decoy or a signaling receptor for VEGF during physiological or pathological angiogenesis. Here we report that VEGF-stimulated nitric oxide (NO) release is inhibited by blockade of VEGFR-1 and that VEGFR-1 via NO negatively regulates of VEGFR-2-mediated proliferation and promotes formation of capillary networks in human umbilical vein endothelial cells (HUVECs). Inhibition of VEGFR-1 in a murine Matrigel angiogenesis assay induced large aneurysm-like structures. VEGF-induced capillary growth over 14 days was inhibited by anti-VEGFR-2-blocking antibody as determined by reduced tube length between capillary connections (P < 0.0001) in an in vitro angiogenesis assay. In contrast, loss of VEGFR-1 activity with a neutralizing anti-VEGFR-1 antibody resulted in an increase in the accumulation of endothelial cells (P < 0.0001) and a dramatic decrease in the number of capillary connections that were restored by the addition of NO donor. Porcine aortic endothelial (PAE) cells expressing human VEGFR-1 but not VEGFR-2 plated on growth factor-reduced Matrigel rearranged into tube-like structures that were prevented by anti-VEGFR-1 antibody or a cGMP inhibitor. VEGF stimulated NO release from VEGFR-1- but not VEGFR-2-transfected endothelial cells and placenta growth factor-1 stimulated NO release in HUVECs. Blockade of VEGFR-1 increased VEGF-mediated HUVEC proliferation that was inhibited by NO donors, and potentiated by NO synthase inhibitors. These data indicate that VEGFR-1 is a signaling receptor that promotes endothelial cell differentiation into vascular tubes, in part by limiting VEGFR-2-mediated endothelial cell proliferation via NO, which seems to be a molecular switch for endothelial cell differentiation.     

Vascular endothelial growth factor receptor-2-mediated mitogenesis is negatively regulated by vascular endothelial growth factor receptor-1 in tumor epithelial cells

Vascular endothelial growth factor (VEGF) receptors are present on nonendothelial cells suggesting that VEGF may mediate nonendothelial effects during organogenesis and tumorigenesis. Here we show that VEGF receptor-1 (VEGFR-1) negatively regulates VEGFR-2-mediated proliferation via nitric oxide (NO) in an epithelial cancer cell line ECV304. Cell proliferation was assessed by [(3)H]thymidine incorporation, fluorescent-activated cell-sorting analysis, and cell number using a Coulter Counter. Total NO generated by the action of nitric oxide synthase was measured by Seivers NOA 280 Nitric Oxide Chemiluminescence Analyser. VEGF (1 ng/ml) stimulated DNA synthesis and increased ECV304 cell number in a manner that was inhibited by a neutralizing anti-VEGFR-2 antibody. In contrast, VEGF (50 ng/ml) stimulated NO release in a manner that was inhibited by functionally neutralizing anti-VEGFR-1 antibody. Blockage of the VEGFR-1 receptor signal with anti-VEGFR-1 stimulated DNA synthesis and increased cell number. Cell-cycle analysis showed that inhibition of VEGFR-1 increased the transition from G(1) to S phase whereas inhibition of VEGFR-2 blocked the VEGF-mediated transition from G(1) to S phase. Finally, the addition of NO donors suppressed both VEGF-mediated proliferation and the increase in growth after blockade of VEGFR-1. Conversely, inhibition of VEGF mediated NO release by nitric oxide synthase inhibitor, L-monomethyl-L-arginine, restored the mitogenic effect of VEGF. These findings identify a dose-dependent reciprocal regulatory mechanism for VEGF via its two receptors. It shows that VEGFR-1 induces cell cytostasis via NO and as such is a suitable target for molecular strategies suppressing tumorigenesis.     

Expression pattern of VEGFR-2 (Quek1) during quail development

The growth and maintenance of the blood and lymphatic vascular systems is to a large extent controlled by members of the vascular endothelial growth factor (VEGF) family via the tyrosine kinase receptors (VEGFRs) expressed in angioblastic cells. Here, we analyzed the Quek1 (VEGFR-2) expression pattern by whole mount in situ hybridization during quail development. During early embryogenesis, Quek1 expression was detected at the caudal end of the blastoderm and primitive streak and in the head paraxial mesoderm. In somites, expression was observed from HH-stage 9 onwards in the dorsolateral region of both the forming and recently formed somites. During somite maturation, expression persists in the lateral portion of the somitic compartments, the dermomyotome and the sclerotome. Additionally, a second expression domain in the maturing somite was observed in the medial part of the sclerotome adjacent to the neural tube. This expression domain extended medially and dorsally and surrounded the neural tube during later stages. In the notochord, expression was observed from HH-stage 23 onwards. In the limb bud, expression was initiated in the mesenchyme at HH-stage 17. During organogenesis, expression was detected in the pharyngeal arches and in the anlagen of the esophagus, trachea, stomach, lungs, liver, heart and gut. Expression was also seen in feather buds from day 7 onwards. Our results confirm the angiogenic potential of the mesoderm and suggest that VEGFR-2 expressing cells represent multiple pools of mesodermal precursors of the hematopoietic and angiopoietic lineages.     

VEGFR-2 as a novel predictor of survival in gastric cancer: A systematic review and meta-analysis

Background

Expression of VEGFRs may affect cancer prognosis. The aim of this work is to evaluate the prognostic significance of VEGFRs of patients with gastric cancer.

Immunolocalization of VEGF, VEGF receptors, EGF-R and Ki-67 in leiomyoma, cellular leiomyoma and leiomyosarcoma

Angiogenic factors, such as vascular endothelial growth factor (VEGF), its receptors and epidermal growth factor receptor (EGF-R), are involved in increased progression in many carcinomas. The aim of this study was to investigate the role of angiogenesis and immunolocalization of VEGF, its receptors, EGF-R and Ki 67 in leiomyomas and leiomyosarcomas using an indirect immunohistochemical method. Samples from patients with leiomyoma, cellular leiomyoma and cellular leiomyosarcoma (n=20 per group) were fixed in 10% formalin and processed using routine paraffin protocols. Following initial histological analysis, samples were immunostained with primary antibodies for VEGF, VEGFR-1, VEGFR-2, EGF-R and Ki-67 using an indirect avidin-biotin peroxidase method. Immunostaining intensities were evaluated as mild, moderate or strong and a semi-quantitative method (H-Score) was used to compare the samples. While mild/moderate EGF-R immunostaining and moderate immunostaining for VEGF and its receptors were observed in samples of leiomyomas, much less immunoreactivity was observed in cellular leiomyomas. All immunoreactivities and immune-stained cells increased in leiomyosarcomas. When scores of intensity and percentage of positive staining cells were compared, all immunoreactivities were shown to be significantly increased in leiomyosarcomas compared to leiomyomas.These results suggest that in leiomyosarcoma, angiogenic factors, such as VEGF, its receptors and EGF-R, may be involved in tumor angiogenesis. Active tumor cells can trigger angiogenesis, interaction with surrounding tissue and in the tissue itself initiating angiogenic activity. Angiogenic growth factors play an important role and induce malignant transformation through both autocrine and paracrine mechanisms. Anti-angiogenic agents may provide a novel therapeutic approach for the treatment of leiomyosarcoma.     

VEGFR-3与肿瘤淋巴转移的研究进展

肿瘤转移通常是人类癌症致死的主要原因之一.淋巴转移是肿瘤特别是癌的常见转移途径,肿瘤细胞转移至淋巴结通常作为肿瘤转移的信号,同时也是患者预后的重要指标.在肿瘤转移过程中,血管内皮生长因子及其受体家族发挥了重要的作用.尤其是淋巴内皮特异性的受体--血管内皮生长因子受体3(VEGFR-3)及其配体VEGF-C/D在促进淋巴管生长具有关键的作用.本文就VEGFR-3与肿瘤淋巴转移的研究进展进行综述.     

血管内皮生长因子C、D和受体3在人胰腺癌组织中的表达

目的:通过观察不同临床指标的人胰腺癌组织VEGF-C、VEGF-D、VEGFR-3的表达,来探讨VEGF-C和VEGF-D对人胰腺癌转移的影响,为癌组织中淋巴管的生成机制以及癌的淋巴道转移机制提供理论依据。方法:取人胰腺癌标本33例及癌旁正常胰腺组织15例,用免疫组化的方法观察VEGF-C、VEGF-D及VEGFR-3在人胰腺癌和癌旁正常胰腺组织中的表达。结果:VEGF-C、VEGF-D和VEGFR-3在胰腺癌组织中的表达比例较在癌旁正常胰腺组织中的表达比例明显增高,并且VEGFR-3的表达与VEGF-D的表达具有显著相关性(P<0.01),与VEGF-C的表达不具有相关性(P>0.05)。胰腺癌组织中VEGF-C和VEGF-D的表达与患者的年龄、性别、远处转移无关(P>0.05)。结论:VEGF-C、VEGF-D在胰腺癌组织中的表达明显增高,并有可能通过与VEGFR-3的结合促进了癌组织中淋巴管的生成,从而对癌的淋巴道转移起促进作用。     

VEGF-C及VEGFR-3在乳腺癌中的表达

目的探讨乳腺癌中血管内皮生长因子-C(VEGF-C)、血管内皮生长因子受体-3(VEGFR-3)的表达及与淋巴管生成的关系。方法采用免疫组化SP法检测57例乳腺癌组织中VEGF-C及VEGFR-3的表达,并在显微镜下记数VEGFR-3标记的脉管。结果淋巴结转移组VEGF-C的阳性率(90.48%)显著高于无淋巴结转移组(47.22%)(P<0.05);淋巴结转移组VEGFR-3阳性脉管数(7.62±1.18)显著高于无淋巴结转移组(5.27±0.96)(P<0.05);VEGF-C的阳性表达与VEGFR-3阳性脉管数呈正相关,相关系数为r为0.882(P<0.05)。结论 VEGF-C及VEGFR-3与乳腺癌的生长,淋巴管的生长及淋巴结的转移有关。     

Expression of the Vascular Endothelial Growth Factor C Receptor VEGFR-3 in Lymphatic Endothelium of the Skin and in Vascular Tumors

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.     

血管内皮生长因子D和血管内皮生长因子受体3在人结肠癌中的表达及淋巴道转移中的作用

目的 观察血管内皮生长因子D(VEGF-D)和血管内皮生长因子受体3(VEGFR-3)在人结肠癌组织中的表达,检测结肠癌组织中的微淋巴管密度(LMVD),探讨VEGF-D和VEGFR-3在淋巴管生成以及结肠癌淋巴道转移中的作用.方法 选择55例不同时期,不同分化程度的人结肠癌组织样本,应用免疫组织化学染色的方法,观察VEGF-D和VEGFR-3在人结肠癌组织中的表达,应用Podoplanin标记淋巴管,检测结肠癌组织中的淋巴管密度.结果 在55例结肠癌组织中,VEGF-D的阳性表达率为54.5%,明显高于在癌周正常组织内的表达(P＜0.05);结肠癌组织中VEGFR-3表达的阳性率为69.1%,明显高于在癌周正常组织内的表达(P＜0.01);并且VEGFR-3的表达与VEGF-D的表达具有显著相关性(P＜0.01).在结肠癌组织中,淋巴结转移阳性组,浸润深度超过肌层组,DukeC、D期的VEGF-D的表达水平和LMVD明显高于淋巴结转移阴性组,浸润深度未超过肌层组,Duke A、B期(P＜0.01),经计数淋巴管数量,癌组织中的LMVD明显高于癌周正常组织(P＜0.01),并且LMVD与VEGF-D的表达显著相关(P＜0.01).结论 结肠癌组织中VEGF-D的表达水平随着癌的浸润和转移程度的增强而增高,并且通过上调其受体VEGFR-3的表达而促进癌组织中淋巴管的生成,从而促进癌的浸润和转移.     

Vasodilation increases pulse pressure variation, mimicking hypovolemic status in rabbits

OBJECTIVE

To test the hypothesis that pulse pressure respiratory variation (PPV) amplification, observed in hypovolemia, can also be observed during sodium nitroprusside (SNP)-induced vasodilation.

INTRODUCTION

PPV is largely used for early identification of cardiac responsiveness, especially when hypovolemia is suspected. PPV results from respiratory variation in transpulmonary blood flow and reflects the left ventricular preload variations during respiratory cycles. Any factor that decreases left ventricular preload can be associated with PPV amplification, as seen in hypovolemia.

METHODS

Ten anesthetized and mechanically ventilated rabbits underwent progressive hypotension by either controlled hemorrhage (Group 1) or intravenous SNP infusion (Group 2). Animals in Group 1 (n = 5) had graded hemorrhage induced at 10% steps until 50% of the total volume was bled. Mean arterial pressure (MAP) steps were registered and assumed as pressure targets to be reached in Group 2. Group 2 (n = 5) was subjected to a progressive SNP infusion to reach similar pressure targets as those defined in Group 1. Heart rate (HR), systolic pressure variation (SPV) and PPV were measured at each MAP step, and the values were compared between the groups.

RESULTS

SPV and PPV were similar between the experimental models in all steps (p > 0.16). SPV increased earlier in Group 2.

CONCLUSION

Both pharmacologic vasodilation and graded hemorrhage induced PPV amplification similar to that observed in hypovolemia, reinforcing the idea that amplified arterial pressure variation does not necessarily represent hypovolemic status but rather potential cardiovascular responsiveness to fluid infusion.     

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1_2-4) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purifi ed (metal affi nity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1_2-4 showed that biotinylated VEGF specifi cally binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fl uorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specifi c receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confi rmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.     

Expression of HIF-1alpha, VEGF and VEGF receptors in the carotid body of chronically hypoxic rat

We examined the protein expression and localization of HIF-1alpha, VEGF, VEGF receptors in the carotid body (CB) of rats breathing 10% inspired oxygen for up to 4 weeks. The immunoreactivity (IR) of HIF-1alpha was distributed numerously in the nuclei of glomus (type-I) and other cells since hypoxia for 1 day, but was faint and scattered in the normoxic CBs. Cytoplasmic staining of the VEGF was intense in glomus cells of the hypoxic but not the normoxic group. The IR levels of HIF-1alpha and VEGF reached plateau at 4 weeks, and the IRs of VEGFR-1 and VEGFR-2 were strongly positive in the hypoxic group. Yet, the expression of VEGFR-1-IR was mild, whereas the VEGFR-2-IR was intense in normoxic CBs, suggesting an upregulation of VEGFR-1 but not VEGFR-2 in hypoxia. Hence, HIF-1 may activate the expression of VEGF and VEGFR-1 in the CB and the expression of VEGF in the chemoreceptors may play a paracrine role in the vascular remodeling during chronic hypoxia.     

人脐带血淋巴管内皮祖细胞的分化及其生物学特征

目的研究脐带血中CD34^＋／CD133^＋／VEGFR-3^＋淋巴管内皮祖细胞经VEGF—C诱导向内皮细胞分化过程中生物学特征的变化,并探讨其分化的机制。方法取脐带血,用PercoU密度梯度离心法分离单形核细胞,再用流式细胞仪分选CD34^＋／CD133^＋／VEGFR-3^＋细胞,然后用VEGF—C诱导分化。在扫描电镜和透射电镜下观察细胞表面形态和细胞内结构的变化,并在激光扫描共焦显微镜下观察特征性标志物的表达变化。结果脐带血中的淋巴管内皮祖细胞表达CD34、CD133和VEGFR-3。CD34^＋／CD133^＋／VEGFR-3^＋细胞经VEGF-C诱导后7d,呈长梭形,细胞伸出板状伪足和丝状伪足,出现较多短的微绒毛。表面可见细胞小凹,细胞质中含有丰富的线粒体和粗面内质网。诱导后14d,细胞已具有内皮细胞的特征,表达淋巴管内皮特异性标志物LYVE-1和5-核苷酸酶,CD133表达消失,细胞质中可见Weibel-Palade小体。结论脐带血中存在CD34^＋／CD133^＋／VEGFR-3^＋淋巴管内皮祖细胞,这些细胞在VEGF—C诱导作用下可能通过VEGF—C／VEGFR-3信号途径分化为淋巴管内皮细胞。     

The phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently immobilized VEGF

Growth factors are a class of signaling proteins that direct cell fate through interaction with cell-surface receptors. Although a myriad of possible cell fates stems from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor – soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm² and 100 pg/cm², respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.