Extent Of Angiogenesis Research Articles

Evaluation of Angiogenesis and Vascular Endothelial Growth Factor Expression in the Bone Marrow of Patients with Aplastic Anemia

It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 +/- 0.7 events per field, versus controls, 5.9 +/- 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 +/- 35 pg/ml versus controls, 444 +/- 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression.

Microvascular density, vascular endothelial growth factor immunoreactivity in tumor cells, vessel diameter and intussusceptive microvascular growth in primary melanoma.

Intussusceptive microvascular growth (IMG) corresponds to one of the types of angiogenesis described in literature. Recent morphological work strongly supports a role for IMG, even during tumor angiogenesis. In this study, the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), the vessel diameter and the IMG have been correlated to the tumor thickness in human primary melanoma specimens. Results showed that an increased microvascular density, a strong VEGF immunoreactivity of tumor cells, a major vessel diameter and a high number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG, are correlated to a high tumor thickness (>3.6 mm). Overall, these data demonstrate for the first time in human primary melanoma a relationship between angiogenesis, VEGF immunoreactivity of tumor cells, vessels diameter and IMG and seem to indicate that VEGF is specifically involved in increasing vessel diameter and IMG.

Microvessel density does not predict outcome in high grade soft tissue sarcoma

The induction of new blood vessel growth into tumours from a pre-existing vascular bed (angiogenesis) is a characteristic of virtually all malignant tumours studied. Previous studies have demonstrated a consistent correlation between the extent of angiogenesis and disease prognosis for a wide range of carcinomas, but not for sarcomas. Aims To investigate the most appropriate method for assessment of microvessel density (MVD) using a cohort of patients with large (>5 cm), deep (intramuscular), high grade soft tissue sarcoma and investigate any relationship between MVD, vascular endothelial growth factor (VEGF) expression and prognosis. Methods Forty-two patients were included in the study. MVD was evaluated in peripheral, central, necrotic and hotspot areas within the tumour by either Chalkley count or total count of immunostained vessels in five high power (×200) visual fields. Results Hot spots of angiogenesis were only present in 33% of specimens. There was a strong correlation p<0.001 between the two methods of quantification with Chalkey method being recommended. VEGF expression evaluated by immunohistochemistry was seen in all but one tumour with strong diffuse cytoplasmic staining within tumour cells, which was not correlated with microvessel density, metastasis or survival. There was a positive correlation between MVD and histological subtype ( p<0.01), and primary tumour size and the development of metastasis ( p=0.049) but MVD was not predictive of metastasis or overall survival. Conclusions Unlike the majority of previous studies involving various types of carcinoma, there was no correlation between microvessel density and metastasis or survival in a group of patients with large (>5 cm), deep, high grade soft tissue sarcoma.

Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization

Clinical observations suggest that genetic factors may influence heterogeneity of angiogenic responses in cardiovascular disease, proliferative diabetic retinopathy, and neoplasia. Experiments among mouse strains using a corneal micropocket assay indicate that extent of angiogenesis may be genetically determined. Here, we established the strain-dependence of hypoxia-induced retinal angiogenesis in multiple mouse strains which paralleled the rank order found for bFGF-induced corneal angiogenesis. Using quantitative real-time RT-PCR, strain-related gene expression differences in retina/choroid between C57BL/6J and 129S3/SvIM, inbred strains with relatively low and high levels of angiogenesis, respectively, after 0, 6, 12, 24, 48, and 96 h hypoxia were determined for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), angiogenic ligands potently induced by hypoxia, and for pigment epithelium-derived factor (PEDF) and thrombospondin-1 (TSP-1), endogenous broad-spectrum antiangiogenic factors. Indirect ELISA was used to correlate VEGF and PEDF protein levels with mRNA expression. At the onset of hypoxia, both PEDF and TSP-1 levels were increased over 15-fold and VEGF was increased over 10-fold compared to Ang-2 in both strains. At the onset of neovascularization (48 h), both VEGF and Ang-2 mRNA levels were increased in the more angiogenic 129S3/SvIM strain (P<0.02), which was not observed among developmental control animals. PEDF expression was higher in the less angiogenic C57BL/6J strain at 6, 12, 24, and 96 h hypoxia (P<0.03), while TSP-1 expression was higher in C57BL/6J throughout the entire time course of hypoxia (4 days) compared to 129S3/SvIM (P<0.02). Among developmental control animals, PEDF and TSP-1 expression was also increased at P14 and P16 in C57BL/6J strain compared to 129S3/SvIM (P<0.02). Strain-dependent expression of both pro- and antiangiogenic growth factors may determine heterogeneity in the angiogenic response and potentially, susceptibility to angiogenesis-dependent diseases.

Loading of a Novel Angiogenic Agent, Ginsenoside Rg1 in an Acellular Biological Tissue for Tissue Regeneration

In this study, the effects of ginsenoside Rg1, a natural compound isolated from Panax ginseng, on human umbilical vein endothelial cell (HUVEC) behavior in vitro, and on angiogenesis and tissue regeneration in genipin-fixed acellular tissue (extracellular matrix, ECM) in vivo, were investigated. Basic fibroblast growth factor (bFGF) was used as a control. The in vitro results indicated that in the presence of bFGF or Rg1, HUVEC proliferation was significantly increased. Both bFGF and Rg1 promoted HUVEC migration in a Transwell plate assay. In addition, bFGF or Rg1 significantly increased the formation of capillary-like network by HUVECs on Matrigel. Thus, both bFGF and Rg1 enhanced multiple components of angiogenic activity in vitro. The in vivo results obtained 1 week postoperatively showed that the extent of angiogenesis in ECMs was significantly enhanced by bFGF or Rg1. At 1 month postoperatively, vascularized neoconnective tissues were found to fill the pores within ECMs loaded with bFGF or Rg1. There was a significant increase in neocapillary density from 1 week to 1 month for ECMs loaded with Rg1, whereas that observed in ECMs loaded with bFGF stayed approximately the same because of the limitations of protein stability. These results suggested that Rg1 may be a new class of angiogenic agent and may be loaded in ECMs to accelerate tissue regeneration.

Angiogenesis in Hodgkin's lymphoma: a morphometric approach in 286 patients with prognostic implications

The significance of angiogenesis in Hodgkin's lymphoma (HL) is not well defined. The aim of this study was to evaluate various morphometric characteristics of microvessels in lymph node sections of 286 patients with HL at diagnosis and investigate their relationship with clinicopathologic parameters and prognosis. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related microvascular parameters were quantitated--after anti-CD34 immunohistochemical staining--in the region of most intense vascularization, using image analysis. An increase in microvessel caliber parameters (area, perimeter, major and minor axis length) and a decrease in MVD were noted with increasing stage. An inverse relationship was recorded between MVD and the number of involved sites (NIS) and LDH. In univariate analysis, overall disease-specific survival was adversely affected by MVD and TVA, whereas inferior failure-free survival (FFS) was associated with the presence of more flattened vessel sections. Multivariate analysis disclosed that the extent of angiogenesis (MVD/TVA), age and the NIS independently affected overall survival. Accordingly, FFS was independently linked to the shape of microvessels and albumin levels or the NIS. In conclusion, our data support the view that angiogenesis in HL provides independent prognostic information, requiring the concomitant evaluation of quantitative and qualitative aspects of microvascular network.

Expression of the proliferation-associated nuclear protein MIB-1 and its relationship with microvascular density in bone marrow biopsies of patients with myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by dysplastic hemopoiesis and an increased risk of leukemic transformation. The process of angiogenesis has been implicated in the pathogenesis and evolution of MDS. In this study the proliferative activity and extent of angiogenesis was examined in bone marrow samples from 54 patients with MDS in relation to clinicopathologic features. Cellular proliferation and microvascular density (MVD) were examined immunohistochemically, using the monoclonal antibody MIB-1 (Ki-67) and an anti-CD34 monoclonal antibody respectively. Serum concentrations of interleukin-6 (IL-6) were measured by ELISA. The results showed that the MIB-1 Labeling Index (MIB-1 LI), MVD and IL-6 increased significantly with advancing severity of disease. Among the MDS-FAB subtypes, MIB-1 LI, MVD and IL-6 were significantly higher in RAEB-t, RAEB and CMML in comparison to RA and RARS (p < 0.0001 in all cases). Similarly, MIB-1 and MVD were increased in patients with score 3 in comparison to scores 0 and 1 in the IPSS system (p < 0.05). All parameters studied were significantly higher in patients versus controls. We conclude that cellular proliferative activity and angiogenesis are associated with disease progression in MDS patients.

A possible role of tryptase in angiogenesis in the brain of mdx mouse, a model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and affects the CNS. Dystrophin is absent in muscle and CNS of both DMD patients and mdx mouse, a model of DMD. While the involvement of vascular compartment in DMD was poorly investigated, some studies suggested a role for mast cells (MC). Tryptase, contained in the MC granules, stimulates angiogenesis in vitro and in vivo. We demonstrated for the first time a correlation between the extent of angiogenesis and the number of tryptase-positive neurons and microvessels and suggest that the tryptase contained in the neurons and in the endothelial cells of the mdx mouse brain may be involved in the regulation of angiogenesis taking place in mdx mouse.

Differential regulation of thrombospondin-1 and thrombospondin-2 after focal cerebral ischemia/reperfusion.

Angiogenesis occurs after cerebral ischemia, and the extent of angiogenesis has been correlated with survival in stroke patients. However, postischemic angiogenesis is short-lived and may be completely terminated within a few weeks after ischemic insult. The molecular mechanism underlying the dissolution of postischemic angiogenic processes is poorly understood. Although the expression of angiogenic genes has been studied in ischemic stroke models, the activation of angiostatic genes after cerebral ischemia has not been investigated. Thrombospondin (TSP)-1 and TSP-2 are naturally occurring angiostatic factors, which inhibit angiogenesis in vivo. The aim of the present study was to explore the expression of TSP-1 and TSP-2 in relation to the evolution of angiogenic process in a focal ischemia model in rats. Rats underwent cortical ischemia in the middle cerebral artery territory for 60 minutes and reperfusion for up to 2 weeks. Northern and Western blot analysis were used to study the temporal profile of TSP-1 and TSP-2 expression at the mRNA and protein level, respectively. In situ hybridization and immunohistochemical studies were used to examine the spatial expression patterns. Double immunostaining was applied to define the cellular origins of TSP-1 and TSP-2. A biphasic expression of TSP-1 was noted after ischemia, peaking at 1 and 72 hours. Endothelial cells in the leptomeninges were the only source of the first TSP-1 peak, whereas endothelial, glial, neuronal, and macrophage cells contributed to the second peak of TSP-1 expression. TSP-2 expression occurred much later and in a monophasic manner, peaking 2 weeks after ischemia. TSP-2 immunoreactivity was observed in endothelial, neuronal, and macrophage, but not glial, cells. TSP-1 was expressed before the peak of angiogenesis, whereas robust TSP-2 expression occurred at the peak of angiogenesis and continued into the period when angiogenesis had completely resolved. Robust expression of TSP-1 and TSP-2, 2 major angiostatic factors, was noted in the ischemic brain with different temporal expression profiles from different cellular origins. The expression of these angiostatic factors, especially TSP-2, likely contributes to the spontaneous resolution of postischemic angiogenesis. Further studies are needed to explore the molecular mechanisms that regulate the balance of angiogenic and angiostatic factors in the ischemic brain.

Revascularization of myocardial scar tissue following prostaglandin E1-therapy in patients with ischemic heart disease.

Prostaglandin E1 (PGE-1) treatment has proved to stimulate angiogenesis in vital non-infarcted myocardium of patients with ischemic cardiomyopathy (ICMP). We investigated infarcted myocardial tissue for a possible angiogenic response to PGE-1. Neovascularization was investigated in infarcted areas of 12 hearts explanted from patients with ICMP who had been treated with PGE-1 before heart transplantation (HTX). In transmural sections containing myocardial scar tissue, CD34 and VEGF were immunohistochemically quantified to estimate capillary density and the extent of angiogenesis. To investigate a possible effect of PGE-1 on collagen turnover, the collagen content was determined in myocardial scar tissue by assessing the intensity of the area positively stained with sirius red. PGE-1-treated patients had significantly more CD34- and VEGF-positive cells in infarcted areas, and showed a significant reduction in collagen content as compared with the non-PGE-1 group (CD34: 120.3 +/- 6.1 vs. 47.7 +/- 6.1 capillary profiles/mm2; VEGF: 52.8 +/- 5.6 vs. 24.0 +/- 4.8 capillary profiles/mm2, and collagen content: 2.18 +/- 0.4 eU vs. 3.59 +/- 0.38 eU). Our data demonstrate that PGE-1 stimulates angiogenesis by upregulating VEGF expression, and reduces fibrosis in cardiac scar tissue of ischemic origin. The induction of therapeutic angiogenesis in vital and at sites of putative dead myocardial scar tissue, along with the hemodynamic improvement in patients with severe ICMP, might explain the favorable clinical outcome in PGE-1-treated patients before HTX.

Effect of venous ulcer exudates on angiogenesis in vitro.

Angiogenesis, the formation of new from existing capillaries, is an important mechanism in venous ulcer healing. The aim of this study was to determine whether venous leg ulcer wound exudates stimulate or inhibit angiogenesis. Fluid exudate was obtained from 16 venous ulcers over a 4-h interval. Five of the ulcers had not healed after more than 1 year of compression bandaging, and five were rapidly healing ulcers. As a control, acute wound fluids were collected from subcutaneous drains in seven patients. Vascular endothelial growth factor (VEGF) at 2 ng/ml acted as a positive control. Tubules stained with an antiendothelial antibody were quantified using an image analysis system. The extent of angiogenesis was expressed as the ratio of the mean tubule length in the test wells over that in blank control wells. Venous ulcer exudates significantly inhibited angiogenesis (mean (95 per cent confidence interval) 0.72 (0.48 to 0.96)) compared with acute wound fluids (2.48 (0.86 to 4.10)) (P < 0.002) and VEGF (1.47 (1.32 to 1.61)) (P = 0.01). Exudates from the five non-healing venous ulcers inhibited angiogenesis (0.31 (0.15 to 0.46)) significantly more than exudates from the five rapidly healing venous ulcers (0.93 (0.21 to 1.65)) (P = 0.03). Fluid exudate from venous ulcers, in particular those that healed slowly, inhibited experimental angiogenesis in this study.

Role of matrix metalloproteinase-9 in angiogenesis caused by ocular infection with herpes simplex virus.

In this report, we demonstrate that herpes simplex virus (HSV) infection of the cornea results in the upregulation of the matrix-degrading metalloproteinase enzyme MMP-9. This enzyme was shown to contribute to the neovascularization process that occurs in the corneal stroma in response to HSV infection. The likely source of MMP-9, at least initially after infection, was neutrophils that were signaled to invade the cornea soon after infection. Corneal infiltrating neutrophils were shown to express MMP-9, and preventing the neutrophil response with specific mAb diminished MMP-9 expression as well as the extent of angiogenesis. Further supporting a role for MMP-9 in HSV-induced corneal angiogenesis was the observation that inhibition of MMP-9 with the specific inhibitor TIMP-1 resulted in reduced angiogenesis. In addition, angiogenesis was diminished in ocularly infected MMP-9 knockout mice. Our results demonstrate that MMP-9 is involved in angiogenesis caused by HSV. Since angiogenesis appears to represent a vital step in the pathogenesis of herpetic stromal keratitis, these results indicate that targeting MMP-9 for inhibition should prove useful for the therapy of herpetic stromal keratitis.

Role of matrix metalloproteinase-9 in angiogenesis caused by ocular infection with herpes simplex virus

In this report, we demonstrate that herpes simplex virus (HSV) infection of the cornea results in the upregulation of the matrix-degrading metalloproteinase enzyme MMP-9. This enzyme was shown to contribute to the neovascularization process that occurs in the corneal stroma in response to HSV infection. The likely source of MMP-9, at least initially after infection, was neutrophils that were signaled to invade the cornea soon after infection. Corneal infiltrating neutrophils were shown to express MMP-9, and preventing the neutrophil response with specific mAb diminished MMP-9 expression as well as the extent of angiogenesis. Further supporting a role for MMP-9 in HSV-induced corneal angiogenesis was the observation that inhibition of MMP-9 with the specific inhibitor TIMP-1 resulted in reduced angiogenesis. In addition, angiogenesis was diminished in ocularly infected MMP-9 knockout mice. Our results demonstrate that MMP-9 is involved in angiogenesis caused by HSV. Since angiogenesis appears to represent a vital step in the pathogenesis of herpetic stromal keratitis, these results indicate that targeting MMP-9 for inhibition should prove useful for the therapy of herpetic stromal keratitis.

Increased angiogenesis in bone marrow of children with acute lymphoblastic leukaemia has no prognostic significance.

The importance of angiogenesis for the growth and viability of solid tumours has been established. Similarly, prognostic information may be gained from the extent of angiogenesis in these tumours. Haematopoietic malignancies should have equal requirements for angiogenesis and important prognostic information may be derived from quantification of bone marrow angiogenic activity. We retrospectively investigated 82 bone marrow trephine biopsies from 41 children with acute lymphoblastic leukaemia (ALL) at diagnosis and following treatment. Nine normal bone marrow trephines from age-matched children were also analysed as controls. The microvessels were stained immunohistochemically with anti-Factor VIII-related antigen (antivWF) and antithrombomodulin (anti-THR). Angiogenesis was quantified manually by two independent observers and was highly reproducible (Pearson's r = 0.91). Staining with anti-vWF and anti-THR was highly specific for microvessels and thetwo stains closely correlated (r = 0.68). Microvessel densities (MVD) at presentation were significantly increased in the majority of patients in comparison with controls (P < 0.0001) and MVD dropped towards normal in remission (P < 0.0001). Of interest, the difference in total vessel counts between leukaemic and normal/remission marrows was contributed solely by small microvessels. There was no significant difference in MVD at presentation or remission from children in poor prognostic groups or those who subsequently relapsed. Similarly, we could not find an association with age, sex, cytogenetic abnormality or disease phenotype.

Chronic hypoxia induces prolonged angiogenesis in skeletal muscles of rat.

Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O(2) for 6 weeks) adult male Wistar rats (final body mass approximately 355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia.

Angiogenesis in patients with craniopharyngiomas: correlation with treatment and outcome.

Craniopharyngiomas are histologically benign epithelial neoplasms of the sellar region that often exhibit aggressive and invasive growth. The authors hypothesized that tumor proliferation, spread, and recurrence are angiogenesis dependent and investigated the significance of vascularization relative to biologic behavior. To the authors' knowledge, angiogenesis for patients with craniopharyngiomas has not been examined to date. The authors measured microvessel densities in resected, histologically proven craniopharyngiomas using immunostains for CD-34, a monoclonal antibody that selectively recognizes endothelial cells. Both histologic types of craniopharyngiomas, adamantinomatous and papillary, were included in the study. In addition, the cellular distribution of vascular endothelial growth factor (VEGF), a strong stimulator of new vessel formation, was assessed by both immunohistochemistry and in situ hybridization for VEGF receptor 2 (VEGFR-2) mRNA expression. Histologically, small numbers of capillaries were identified in temporal stroma but not in their epithelial components. Immunohistochemistry revealed strong, conclusive cytoplasmic immunoreactivity for VEGF in the epithelial cells of both adamantinomatous craniopharyngiomas and papillary craniopharyngiomas. In situ hybridization showed that VEGFR-2 mRNA was expressed widely, not only in neoplastic epithelium but also in capillary endothelium. Tumors with greater microvessel density regrow more frequently compared with tumors that have lower microvessel density, suggesting that the extent of angiogenesis is of prognostic value in patients with craniopharyngioma. VEGFR-2 may act as a key modulator of VEGF activity in endothelial cells and nonendothelial cells, indicating that VEGF plays an important role in the behavior of craniopharyngiomas.

Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with progression in human neuroblastoma

In human tumors changes in angiogenesis and expression of extracellular matrix-degrading enzymes occur simultaneously during invasion and metastasis. Tissues from 20 biopsies of human neuroblastoma (NB) were investigated immunohistochemically by using an antibody against factor VIII to determine their microvessel number, and by in situ hybridisation to determine the expression of mRNA of the matrix metalloproteinase-2 (MMP-2) and MMP-9. The extent of angiogenesis and the expression of the MMP-2 and MMP-9 mRNA were upregulated in advancing stages. These in situ data suggest that angiogenesis and degradation of extracellular matrix occur simultaneously with NB tumor progression.

Regulation of neoplastic angiogenesis.

The progressive growth of neoplasms and the production of metastasis depend on the development of adequate vasculature, i.e., angiogenesis. The extent of angiogenesis is determined by the balance between positive- and negative-regulating molecules that are released by tumor and host cells in the microenvironment. The growth of many neoplasms is associated with the absence of the endogenous inhibitor of angiogenesis, interferon beta (IFN beta). A survey of multiple mouse and human tumors shows a lack of IFN beta associated with extensive angiogenesis. Therapy with IFN alpha or beta either by subcutaneous injection of the protein or by introduction of viral vectors that contain the IFN beta gene inhibit angiogenesis and, hence, progressive tumor growth.

Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia.

Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, including peripheral arterial occlusive disease (PAOD). Because angiogenesis and collateral vessel formation are important self-salvage mechanisms for ischemic PAOD, we examined whether HH modulates angiogenesis in vivo in a rat model of hindlimb ischemia. Rats were divided into 3 groups: the control group was given tap water, the HH group was given water containing L-methionine (1 g x kg(-1) x d(-1)), and the HH+L-arg group was given water containing methionine (1 g x kg(-1) x d(-1)) and l-arginine (2.25 vol%). At day 14 of the dietary modifications, the left femoral artery and vein were excised, and the extent of angiogenesis and collateral vessels in the ischemic limb were examined for 4 weeks. Plasma homocysteine levels significantly increased (P:<0.001), and plasma and tissue contents of nitrite+nitrate as well as tissue cGMP levels significantly decreased in the HH group compared with the control group (P:<0.01). Laser Doppler blood flowmetry (LDBF) revealed a significant decrease in the ischemic/normal limb LDBF ratio in the HH group at days 7, 14, 21, and 28 (P:<0.01 versus control). Angiography revealed a significant decrease in the angiographic score in the HH group at day 14 (P:<0.001 versus control). Immunohistochemistry of ischemic tissue sections showed a significant reduction in the capillary density in the HH group (P:<0. 001 versus control). Oral l-arginine supplementation in rats with HH (HH+L-arg) restored the decreased plasma and tissue nitrite+nitrate and cGMP contents (P:<0.05) as well as angiogenesis, as assessed by LDBF (P:<0.05 versus HH), angiographic score (P:<0.01 versus HH), and capillary density (P:<0.001 versus HH). In summary, HH impaired ischemia-induced angiogenesis and collateral vessel formation in a rat model of hindlimb ischemia in vivo. The mechanism of the HH-induced impairment of angiogenesis might be mediated in part by a reduced bioactivity of endogenous NO in the HH state.

Increased Angiogenesis in the Uterine Cervix Associated with Human Papillomavirus Infection

We studied the relationship between angiogenesis (using the CD34 antibody), the presence of human papilloma virus (HPV) infection, HPV E6 protein expression and the accumulation of p53 protein at various phases of tumour progression in the uterine cervix. Expression of CD34, p53 and HPV E6 protein was evaluated by immunocytochemistry. Presence of the mutant p53 was detected using a mutant specific ELISA, and the type of HPV was determined by the Polymerase Chain Reaction. A total of 230 cervical tissue samples were analyzed and included 40 cases of apparently normal cervical epithelium, 37 low grade squamous intraepithelial lesions (SILs), 43 high grade SILs, 36 well-differentiated squamous cell carcinomas (DSCC), 31 moderately differentiated (MDSCC) and 43 poorly differentiated carcinomas (PDSCC). There was an excellent correlation between the extent of angiogenesis and histological abnormality (r = 0.912, p = 0.000004). The least extent of angiogenesis was seen in normal cervical tissue and low grade SILs where the mean (low power) intra lesional vascular density (ILVD) was 12 +/- 1.13 and 25.66 +/- 5.20, respectively. In high grade squamous intraepithelial lesions (SILs), the mean ILVD value was 80.84 +/- 25.57. In well-differentiated squamous cell carcinomas (WDSCC's) the mean value was 144.22 +/- 28.67 while in moderately differentiated squamous cell carcinomas (MDSCC's) the mean value was 166.29 +/- 34.95 and in poorly differentiated tumours (PDSCC's) 192.42 +/- 27.98. The extent of angiogenesis also correlated to presence of HPV (r = 0.505, p = 0.00001). Increased CD34 expression was associated with the presence of HPV types 16 and 18. A similar correlation was also evident in HPV, 16/18 infected cases expressing the E6 protein (r = 0.612, p = 0.000001). CD34 expression also correlated well with p53 accumulation (r = 0.859, p = 0.000002). Presence of HPV infection significantly correlated with the extent of histological abnormality (r = 0.467, p = 0.00001). Expression of E6 also showed this significant correlation (r = 0.644, p = 0.00002). Accumulation of p53 was significantly more elevated in HPV 16-infected lesions (r = 0.518, p = 0.00001) and E6-expressing cells (r = 0.650, p = 0.000004). Only 12 of the 230 cases analyzed showed presence of the mutant p53 protein. Angiogenesis appears to increase with histological abnormality in the uterine cervix. Angiogenesis also appears to be influenced by high risk HPV infection, the expression of the E6 transforming protein and the p53 tumour suppressor protein.