Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants

Crosslinked hyaluronan (HA) hydrogels preloaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), were employed to elicit new microvessel growth in vivo. As a major glycosaminoglycan (GAG) component of extracellular matrix (ECM), HA is an excellent biopolymeric building block for new biomimetic, biocompatible therapeutic materials. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Histologic analysis showed that each of the groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.001). Treatment groups receiving either co-delivery of both KGF and VEGF, an HA hydrogel lacking a growth factor or HA hydrogels containing a single cytokine were statistically unchanged with time, whereas treatment with KGF alone produced continuing increases in vascularization from day 7 to day 14. Strikingly, presentation of both VEGF and KGF in crosslinked HA generated intact microvessel beds with well-defined borders. In addition, an additive response to co-delivery of both cytokines in the HA hydrogel was observed. The HA hydrogels containing KGF+VEGF produced the greatest angiogenic response of any treatment group tested (NI=5.4 at day 14, where NI is a neovascularization index). This was 33% greater vessel density than in the next largest treatment group, that received HA+KGF (NI=4.0, p<0.002). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and KGF from crosslinked HA films.     

Stimulation of in vivo angiogenesis using dual growth factor-loaded crosslinked glycosaminoglycan hydrogels

Crosslinked, chemically modified hyaluronan (HA) hydrogels pre-loaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), were employed to elicit new microvessel growth in vivo, in both the presence and absence of heparin (Hp) in the gels. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Analysis of neovascularization showed that each of the treatment groups receiving an implant, except for HA/Hp at day 14, demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.015). Treatment groups receiving either Ang-1 alone, or aqueous co-delivery of both Ang-1 and VEGF, were statistically unchanged with time. In contrast, film delivery of both growth factors produced continuing increases in vascularization from day 7 to day 14 in the absence of Hp, but decreases in its presence. However, presentation of both VEGF and Ang-1 in crosslinked HA gels containing Hp generated intact microvessel beds with well-defined borders. The HA hydrogels containing Ang-1+VEGF produced the greatest angiogenic response of any treatment group tested at day 14 (NI=7.44 in the absence of Hp and 4.67 in its presence, where NI is a neovascularization index). Even in the presence of Hp, this had 29% greater vessel density than the next largest treatment group receiving HA/Hp+VEGF (NI=3.61, p=0.04). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and Ang-1 from crosslinked HA films.     

The correlation of angiogenesis with metastasis in primary cutaneous melanoma: a comparative analysis of microvessel density, expression of vascular endothelial growth factor and basic fibroblastic growth factor

AIM: To establish whether there is a correlation between angiogenesis and metastasis in primary cutaneous melanoma (PCMM). METHODS: We studied the microvessel density and the expression of vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) in 22 cases of PCMM with metastasis at presentation (metastatic group) and 28 cases of PCMM without metastasis for 24 months or more (non-metastatic group). Microvessels were stained with CD31/PECAM-1 antibody and counted. We assessed the proportion of VEGF expression in tumour cells, lymphocytes infiltrating the tumour (TIL) and lymphocytes at the periphery of the tumour, as well as the proportion of bFGF expression in tumour cell cytoplasms, nuclei and intra- and peritumoral vessels. RESULTS: An increased microvessel density was detected in the metastatic group (15-33 [24.09 +/- 5.55] versus 2-24 [12.96 +/- 6.02]). Moreover, enhanced expression of VEGF in tumour cells and peritumoral lymphocytes (Chi-square p = 0.038 and p = 0.018) and bFGF in peritumoral vessels (chi(2) p = 0.013) correlated with the simultaneous presence of melanoma metastasis in PCMM. Furthermore, microvessel density was correlated with the expression of bFGF in peritumoral vessels (rs = 0.53, p = 0.049) and VEGF in tumour cells (rs = 0.37, p = 0.019). CONCLUSION: Microvessel density as well as the expression of both VEGF and bFGF might be informative concerning the progression of melanoma.     

Differential Endothelial Migration and Proliferation to Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor

Abstract

Neovascularization is a feature of a variety of pathological processes. We compared the characteristics of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on migration and proliferation of human umbilical vein endothelium (HUVEC). Both VEGF and bFGF induced endothelial cell migration at similar concentrations (½ max. VEGF = ～1.0 ng/ml, bFGF = ～5.0 ng/ml). However, VEGF-stimulated migration was two-fold greater than bFGF at 1 and 10 ng/ml (p < 0.05). In contrast, bFGF induced proliferation four-fold more effectively than VEGF (½ max. 1 ng/ml and 1.4 ng/ ml respectively). Checkerboard migration assays for bFGF showed a predominantly chemokinetic pattern, whereas VEGF was predominantly chemotactic. VEGF and bFGF were not synergistic in monolayer proliferation and migration assays. Three angiogenesis inhibitors, alpha-interferon, TNP-470, and platelet factor-4, inhibited VEGF and bFGF induced cell migration. These results indicate that VEGF and bFGF are chemoattractants that stimulate endothelial migration by different mechanisms and that both can be inhibited by known angiogenesis inhibitors.     

Vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF) play a central role in the pathogenesis of digital clubbing

Digital clubbing is associated with many unrelated serious diseases but its pathogenesis remains a clinical enigma. It has been hypothesized that platelet clusters impacting in the distal vasculature mediate the morphological changes of clubbing. Since the multifunctional cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are released on platelet aggregation and are hypoxically regulated, the present study has examined their role in clubbing using immunohistochemistry. Basic fibroblast growth factor (bFGF), transforming growth factor-beta 1 (TGF-beta1), microvessel density, carbonic anhydrase IX (CAIX), hypoxia inducible factor (HIF)-1alpha, and HIF-2alpha were also measured. There was a significant increase in VEGF (p = 0.01), pKDR (p = 0.03), PDGF (p = 0.017), and HIF-1alpha and HIF-2alpha (p = 0.004 and p = 0.004, respectively) expression together with a significant increase in microvessel density (p = 0.03) in the stroma in clubbed digits compared with controls. There was no difference in CAIX (p = 0.25), TGF-beta1 (p = 0.66) or bFGF (p = 0.18) between affected and control groups. These findings suggest that VEGF and PDGF are released after platelet impaction and that their expression is hypoxically enhanced in the stroma after capillary occlusion. VEGF may synergize with PDGF in inducing the stromal and vascular changes present in digital clubbing.     

Effect of EGF and bFGF on Fibroblast Proliferation and Angiogenic Cytokine Production from Cultured Dermal Substitutes

Abstract

Growth factors accelerate wound healing but the underlying mechanisms remain poorly understood. The aim of this study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on fibroblast proliferation and production of angiogenic factors from cultured dermal substitutes (CDS). In the first experiment, fibroblasts were seeded into a flask at a density of 1 × 10⁴ cells/cm².Cell proliferation was assessed after culturing in media containing EGF or bFGF at concentrations ranging from 2 to 50 μg. The number of fibroblasts increased significantly in the presence of EGF or bFGF, but fibroblasts detached from the flasks in the presence of 50 μg bFGF. In the second experiment, CDS were prepared by incorporating fibroblasts into collagen gels. To make a wound surface model, the CDS was elevated to the air–liquid interface, on which a spongy sheet of hyaluronic acid (HA) containing EGF or bFGF was placed. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS after 1 week of cultivation was measured by ELISA. When the CDS was covered with a HA sponge containing EGF (Group 1), fibroblasts released 3.5-times more VEGF compared with a HA-alone sponge (control group). When covered with a HA sponge containing bFGF (Group 2), 8.7-times more VEGF was released compared with the control group. Fibroblasts in Groups 1 and 2 released 9.6- and 9.3-times more HGF, respectively, compared with the control group. Thus, EGF stimulates fibroblasts to produce VEGF and HGF, in addition to its ability to enhance epidermal cell proliferation.     

Hypercholesterolemia impairs angiogenesis in patients with breast carcinoma and, therefore, lowers the risk of metastases

Our aim was to study the effect of hypercholesterolemia on angiogenesis induced by breast carcinoma. Of 51 patients with invasive ductal carcinoma, 28 had hypercholesterolemia and 23 had normocholesterolemia. The intratumoral microvessel density (MVD) was evaluated by using anti-CD31 antibody. The expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on endothelial and tumor cells was examined and graded semiquantitatively. Patients with normocholesterolemia had a higher MVD (76.4 +/- 8.2) than those with hypercholesterolemia (54.6 +/- 5.1) (P < .01). The risks of recurrence and distant metastasis were higher in patients with normocholesterolemia than in patients with hypercholesterolemia (P < .01). Patients with hypercholesterolemia showed lower expression of endothelial VEGF and bFGF than patients with normocholesterolemia (P < .05 and P < .01, respectively). In addition, tumoral bFGF and VEGF expression showed negative correlation with the presence of hypercholesterolemia (P < .01). We suggest that hypercholesterolemia impairs angiogenesis by suppressing endothelial and tumoral bFGF and VEGF expression and, therefore, lowers the risk of metastases in cases of invasive breast carcinoma.     

Coexpression of heparanase, basic fibroblast growth factor and vascular endothelial growth factor in human esophageal carcinomas

Heparan sulfate (HS), which is degraded by heparanase, plays an important role in cell adhesion, insolubility of the extracellular matrix (ECM) and as a reservoir for various growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). In the present study, we examined the immunohistochemical expression of heparanase, bFGF and VEGF, and evaluated the correlation between their expression and microvessel density (MVD) in human esophageal carcinomas. Heparanase, bFGF and VEGF were immunolocalized predominantly to the carcinoma cells, but they were also localized to the endothelial cells of microvessels near the carcinoma cell nests. In carcinomas with invasion of the muscular layer or adventitia, heparanase staining was stronger at the invasive areas of carcinomas than the intraepithelial spread. Expression of heparanase and bFGF and the degree of MVD were associated with tumor invasion, lymph node metastasis and pathological stages. Cases with positive staining for heparanase, bFGF or VEGF tended to have a higher MVD than those without staining, and carcinomas with concomitant expression of heparanase, bFGF and VEGF showed the highest MVD. The level of heparanase mRNA expression was directly correlated with the MVD. In addition, heparanase-positive cases had a higher positive ratio of bFGF and VEGF compared with the heparanase-negative cases. These data suggest the possibility that heparanase may contribute to not only cancer cell invasion but also angiogenesis probably through degradation of HS in the ECM and release of bFGF and VEGF from the HS-containing ECM.     

Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are produced at sites of inflammation. Previously, we demonstrated that bFGF enhances leukocyte recruitment and endothelial cell adhesion molecule (CAM) expression during inflammation. Here, we investigated the influence of VEGF during acute inflammation and whether VEGF and bFGF cooperate to modulate leukocyte recruitment. Inflammation was induced in skin of rats by intradermal injection of inflammatory stimuli +/- VEGF +/- bFGF. Migration of 51Cr-monocytes and 111In-polymorphonuclear leukocytes (PMN) to the dermal lesions and 125I-anti-CAM monoclonal antibody binding to the dermal vasculature were quantitated after 2 h. VEGF significantly enhanced tumor necrosis factor alpha (TNF-alpha)-induced monocyte recruitment by 39 +/- 16% and increased P-selectin, E-selectin, and intercellular CAM-1 expression by two- to threefold over TNF-alpha alone. However, recruitment of monocytes to TNF-alpha + interferon-gamma (IFN-gamma) and of PMN to all stimuli tested was not affected by VEGF. In contrast, bFGF enhanced recruitment of both leukocyte types to all stimuli tested. With the potent TNF-alpha + IFN-gamma stimulus, in contrast to bFGF, VEGF did not enhance E-selectin or ICAM-1 expression. bFGF, but not VEGF, increased the chemotactic activity for PMN in TNF-alpha + IFN-gamma-inflamed sites by 54%. The limited effect of VEGF on these mechanisms likely contributed to the differential effect of VEGF and bFGF on leukocyte recruitment. However, VEGF + bFGF increased PMN recruitment more than did either factor alone. Thus, bFGF and VEGF differentially but synergistically enhance leukocyte recruitment to inflammatory stimuli and individually as well as jointly function as positive regulators of inflammatory cell recruitment.     

VEGF、bFGF、TGFβ1在非小细胞肺癌中的表达及临床意义

目的 探讨VEGF、bFGF、TGFβ1在非小细胞肺癌的表达及与其生物学行为特征的关系.方法 应用链霉菌抗生素蛋白-过氧化物酶连结(streptavidin-perosidase,SP)免疫组化方法检测VEGF、bFGF、TGFβ1及CD34在50例非小细胞肺癌的表达,并进行微血管密度计数,以18例癌旁组织为对照.结果 VEGF、bFGF、TGFβ1的阳性表达率分别为68%、58%、60%,VEGF、bFGF、TGFβ1表达均与NSCLC的临床分期(χ2值分别为4.535、3.864、3.852,P＜0.05)、淋巴结转移有关(χ2值分别为4.235、3.876、4.151,P＜0.05),而TGFβ1,表达还与组织学类型有关(χ2=4.443,P＜0.05),鳞癌比腺癌表达率高.MVD表达与NSCLC的临床分期、淋巴结转移有关(t值分别为2.312和2.865,P＜0.05),与组织学类型、组织分化程度无关;且四者表达在癌组织与癌旁组织之间均有显著性统计学意义.VEGF、bFGF表达与MVD有关(t值分别为2.518和3.679,P＜0.05);TGFB.表达与MVD无显著关系.结论 VEGF、bFGF、TGFβ1在NSCLC均有很好的表达,并且其表达与NSCLC的生长、进展、转移等生物学关系密切;VEGF、bFGF在肿瘤血管形成、转移中起重要作用,TGFβ1可能与机体免疫功能降低及肿瘤细胞免疫逃逸有关.     

Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma

We examined the expression levels of a number of metastasis-related genes to determine the relationship of these levels to the development of metastasis in renal cell carcinoma. Gene expression was examined in 46 formalin-fixed, paraffin-embedded, archival specimens of primary organ-confined, clear-cell, renal cell carcinoma from patients who had undergone radical nephrectomy. Twenty samples were from patients who did not have metastasis after a median of 48 months; 26 were from patients with either synchronous or metachronous metastases. Microvessel density was assessed by anti-CD-34 immunohistochemical analysis. The expression levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), matrix metalloproteinases (MMP)-2 and -9, and E-cadherin were examined at the periphery of the tumor by a colorimetric in situ mRNA. The expression levels of bFGF, VEGF, IL-8, MMP-2, and MMP-9 were significantly higher in primary renal tumors from patients with either synchronous or metachronous metastases than those who were disease-free at a median of 48 months of follow-up. Multivariate analysis of disease-free survival showed that the ratio of MMP-9 to E-cadherin (P = 0.012) and the expression level of bFGF expression (P = 0.045), were independent predictors for the development of metastases. The expression levels of bFGF, VEGF, and IL-8 did not correlate with microvessel density, which in itself was not a significant predictor of progression (P = 0.21). In summary, expression levels of genes that regulate metastasis angiogenesis can predict the metastatic potential in individual patients with organ-confined clear-cell renal carcinoma.     

Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF

Controlled release of human vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate growth factor (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (15 kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel growth at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.     

Expression of angiogenic factors and apoptotic factors in leiomyosarcoma and leiomyoma.

Angiogenesis is essential for tumor growth and metastasis. Some angiogenic factors, such as vascular endothelial growth factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), transforming growth factor-alpha (TGF-alpha) and basic fibroblast growth factor (bFGF) are involved in increased angiogenic activity and disease progression in many carcinomas. However, there is little information regarding the association between angiogenic factors and leiomyosarcoma. Although there are abundant vessels in the sarcoma which enable it to easily receive nutrition and medicinal components, chemotherapy cannot effectively treat leiomyosarcoma. This means the resistance to anticancer drugs in leiomyosarcoma is very strong. However, the resistant mechanism is still unclear. In this study, expressions of VEGF, PD-ECGF, TGF-alpha, bFGF, intratumoral microvessel density (IMVD), and p53, Bcl-2 and Bax were examined by immunohistochemistry in 30 patients with leiomyosarcoma and 21 patients with leiomyoma. With regard to angiogenesis, PD-ECGF and TGF-alpha were closely associated with an increase in IMVD (p=0.012, 0.0196, respectively), and VEGF and PD-ECGF were significantly expressed in leiomyosarcoma compared with leiomyoma (p=0.041, 0.041, respectively). Although p53 expression in leiomyosarcoma was significantly higher than in leiomyoma (p=0.016), the frequency of p53 positivity was not so high (47%). On the other hand, the ratio of Bcl-2/Bax in leiomyosarcoma was significantly higher than that in leiomyoma (p=0.033). The findings of this study suggest that in leiomyosarcoma, angiogenic factors, such as PD-ECGF, VEGF and TGF-alpha expression may be involved in tumor angiogenesis, and the frequently high ratio of Bcl-2/Bax and expression of p53 gene mutation might be related to chemoresistance mechanism.     

Biomaterial mesh seeded with vascular remnants from a quail embryo has a significant and fast vascular templating effect on host implant tissue

Seeding biomaterial implants with vascular remnants has the potential to facilitate host vessel ingrowth via a vascular templating effect. Vessels from quail embryo were grown into a polyurethane fibroporous mesh and the samples were frozen-thawed and then implanted in rat subcutaneous dorsum. Results show that the process of revascularization, using the quail vessel remnants, occurred over the first 3 days after implantation and resulted in functional vessels. Rat endothelial cells were found in the quail templates on day 1. On day 2 the endothelial cells formed a confluent layer and started producing laminin. By this time approximately 70% of the rat vessel tissue in the implant had grown into quail vascular remnants, indicating that the quail vessels were extensively used as templates for host vessel ingrowth. Laminin production was increased and collagen production started by day 3, at which time the vessels were functional in that rat blood flowed through them. At 2 weeks host vessel density was approximately twice that of control samples; thus the implant substantially enhanced the size of the vascular network. For meshes that additionally received vascular endothelial growth factor (VEGF) seeding before implantation, vessel density at 2 weeks was enhanced over samples with quail embryo alone. However, the quail was found to have the greatest angiogenic effect above any of the implant components-quail, VEGF, and collagen. Tissue engineering of vessel templates may thus be a realistic solution to effective fast vascularization of biomaterials.     

Prognostic impact of VEGF, CD31, CD34, and CD105 expression and tumour vessel invasion after radical surgery for IB-IIA non-small cell lung cancer

AIMS: To evaluate the prognostic impact of tumour angiogenesis assessed by vascular endothelial growth factor (VEGF), microvessel density (MVD), and tumour vessel invasion in patients who had undergone radical resection for stage IB-IIA non-small cell lung cancer (NSCLC). METHODS: Fifty one patients (42 men, nine women; mean age, 62.3 years; SD, 6.9) undergoing complete surgical resection (35 lobectomy, 16 pneumonectomy) of pathological stage IB (n = 43) and IIA (n = 8) NSCLC were evaluated retrospectively. No patient underwent postoperative chemotherapy or neoadjuvant treatment. Tumour specimens were stained for VEGF and specific MVD markers: CD31, CD34, and CD105. RESULTS: VEGF expression significantly correlated with high CD105 expression (p < 0.0001) and tumour vessel invasion (p = 0.04). Univariate analysis showed that those patients with VEGF overexpression (p = 0.0029), high MVD by CD34 (p = 0.0081), high MVD by CD105 (p = 0.0261), and tumour vessel invasion (p = 0.0245) have a shorter overall survival. Furthermore, multivariate Cox regression analysis showed that MVD by CD34 (p = 0.007), tumour vessel invasion (p = 0.024), and VEGF expression (p = 0.042) were significant predictive factors for overall survival. Finally, the presence of both risk factors, tumour vessel invasion and MVD by CD34, was highly predictive of poor outcome (odds ratio, 3.4; 95% confidence interval, 1.7 to 6.5; p = 0.0002). CONCLUSIONS: High MVD by CD34 and tumour vessel invasion are more closely related to poor survival than the other neoangiogenetic factors in stage IB-IIA NSCLC. This may be because these factors are more closely related to the metastatic process.     

Tumour proliferation, angiogenesis, and ploidy status in human colon cancer

AIMS: Tumour angiogenesis is essential for carcinogenesis and facilitates the process of tumour development and metastasis. Vascular endothelial growth factor (VEGF) is a well characterised angiogenetic factor and is known to play a crucial role in new vessel development. To gain further insight into the effects of microvessel density and VEGF expression in colon cancer, their relation with tumour proliferation, ploidy status, and p53 expression was investigated in colon cancer. METHODS: Tissue samples of 50 archived colon cancers were analysed by immunohistochemistry for VEGF, p53, and the endothelial cell marker, von Willebrand factor (VWF), using specific antibodies. The same samples were re-cut for flow cytometric studies to obtain S phase fraction (SPF) and ploidy status. RESULTS: A positive significant correlation was found between SPF and angiogenesis. The median microvessel count in high SPF tumours was significantly higher than in low SPF ones. No association was found between VEGF expression and SPF. A positive correlation was found between ploidy status and p53 expression and microvessel count. Furthermore, a positive correlation was established between DNA ploidy, VEGF expression, and microvessel count. CONCLUSION: This study provides evidence that in colon cancer, tumour growth may be stimulated by vascular supply, and the lack of a correlation between tumour cell proliferation and VEGF expression indicates that these two parameters may be regulated by separate mechanisms. Furthermore, the positive correlation between microvessel density, VEGF expression, and ploidy status provides more evidence that genetic alterations are involved in tumour angiogenesis.     

Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis

BACKGROUND: Angiogenesis is a prerequisite for airway remodeling in bronchial asthma. Several growth factors may play important roles in inflammation and angiogenesis through effects on inflammatory cell infiltration or neovascularization. OBJECTIVE: We sought to compare bronchial vascularity and expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin in bronchial biopsy specimens from asthmatic and healthy control subjects. METHODS: Bronchial biopsy specimens were obtained from 16 asthmatic subjects and 9 normal control subjects. The number of vessel profiles and the vascular area per unit area on a histologic section were estimated by using computerized image analysis after staining for type IV collagen in vessel walls. Numbers of VEGF+, bFGF+, and angiogenin+ cells were determined by means of immunoreactivity. RESULTS: The airways of asthmatic subjects had significantly more vessels (P < .05) and greater vascular area (P < .001) than that observed in control subjects. Asthmatic subjects exhibited higher VEGF and bFGF and angiogenin immunoreactivity in the submucosa than did control subjects (P < .001, respectively). Significant correlations were detected between the vascular area and the numbers of angiogenic factor-positive cells (VEGF: rs = 0.93, P < .001; bFGF: rs = 0.83, P < .001; angiogenin: rs = 0.88, P < .001) within the asthmatic airways. Furthermore, the degree of vascularity was inversely correlated with airway caliber and airway responsiveness. Colocalization analysis revealed that the angiogenic factor-positive cells were CD34+ cells, eosinophils, and macrophages. CONCLUSION: Our results suggest that increased vascularity of the bronchial mucosa in asthmatic subjects is closely related to the expression of angiogenic factors, which may then contribute to the pathogenesis of asthma.     

Expression of thymosin beta10 and its role in non-small cell lung cancer

The exact role of thymosin beta10 in lung cancer progression remains unclear. We investigated by immunohistochemistry the expression of thymosin beta10 protein in tumors and tumor-adjacent tissues from 69 patients with non-small cell lung cancer. The relationship of thymosin beta10 expression with vascular endothelial growth factor, vascular endothelial growth factor-C, microvessel density, and lymphatic vessel density was determined; clinicopathologic factors and surgical treatment outcome were also studied. The results showed that thymosin beta10 was mainly expressed in the cytoplasm of lung cancer cells, and the overexpression of thymosin beta10 was correlated with advanced clinical stage (P = .026), distant metastases (P = .016), lymph node metastases (P = .007), poor degree of differentiation (P = .03), and poor postoperative survival (P = .004). Furthermore, thymosin beta10 overexpression was associated with vascular endothelial growth factor (P = .004), vascular endothelial growth factor-C (P = .017), microvessel density (P = .000), and lymphatic vessel density (P = .002). The lowest survival rate was observed in the patients with high thymosin beta10, positive vascular endothelial growth factor, and high microvessel density (P = .007) or in the patients with high thymosin beta10, positive vascular endothelial growth factor-C, and high lymphatic vessel density (P = .005). These results suggest that thymosin beta10 might induce microvascular and lymphatic vessel formation by up-regulating vascular endothelial growth factor and vascular endothelial growth factor-C in lung cancer tissues, thus promoting the distant and lymph node metastases and being implicated in the progression of non-small cell lung cancer.     

兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

建立体外分离、培养及鉴定兔骨髓血内皮祖细胞（endothelial progenitor cell,EPCs）的方法,并探讨其在血管组织工程构建过程中的功能。采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被（FN）的组织工程血管支架上,加入血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析。分离培养的骨髓单个核细胞呈典型的＂铺路石样＂外观。经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞;种植细胞10 d后结果显示：加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞。HE染色显示：内皮细胞在血管支架上成活并较为均匀;免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34。兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）和纤维连接蛋白（FN）的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件。     

Expression of vascular endothelial growth factor in diffuse malignant pleural mesothelioma

Angiogenesis is an important part of normal and pathological processes, including tumour growth, metastasis, inflammation and wound healing. VEGF is the best known angiogenic factor, implicated in tumour-associated microvascular hyperpermeability and carcinogenesis. We investigated 103 malignant pleural mesotheliomas, analysing the expression of vascular endothelial growth factor using immunohistochemistry and insitu hybridization. The grade of microvessel density was assessed with the aid of anti-factor-VIII antibodies. An increased expression of VEGF was found in biphasic and epithelioid mesotheliomas, correlating in a statistically significant manner (P<0.042). Insitu hybridization confirmed the specificity of VEGF mRNA expression. There was a robust correlation between VEGF expression and increased microvessel density (P<0.001), and positive mesotheliomas had significantly higher microvessel densities than negative specimens. There was also a significant correlation between microvessel density and histological pattern. As growth pattern tended towards biphasic and sarcomatoid mesotheliomas the density of micovessels decreased (P<0.05).