alpha-Cell neogenesis in an animal model of IDDM

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of beta-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.     

Insulin resistance and impaired insulin secretion due to phosphofructo-1-kinase-deficiency in humans

Recent epidemiologic evidence suggests that patients with chronic pancreatitis (CP) have an increased risk of developing pancreatic carcinoma (PCA). In spite of numerous similarities in both diseases, mechanisms for progression from CP to PCA are poorly understood. We hypothesized that enhanced angiogenesis might play a pivotal role in the etiology and histopathology of both CP and PCA, and thus form a possible link between precancer and carcinoma. In surgical specimens of 18 patients with CP, 10 with PCA, and 18 controls, absolute numbers of blood vessels and relative blood vessel density were assessed after immunostaining of endothelial cells for von Willebrand factor and PECAM-1 (platelet/ endothelial cell adhesion molecule-1). Furthermore, the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and of VEGF (vascular endothelial growth factor) was investigated in all specimens. Both CP and PCA exhibited areas of high vascular density ("hot spots"). The mean number of blood vessels in these areas in PCA was 132.2+/-16.8 per mm2, and in CP, 99.2+/-7.4 per mm2. The mean vessel count in controls was 25.1+/-5.1. Relative vessel density was increased in both PCA (41.3+/-3.5%) and CP (30.6+/-2.6%) versus controls (8.0+/-0.8%). Both absolute vessel count and relative vessel density were significantly higher (p<0.05) in PCA than in CP. Enhanced expression of ICAM-1 in CP and PCA was seen in ductal cells in CP and cancer cells. In controls, ICAM-1 and VCAM-1 were expressed only at low levels in endothelial cells. VCAM-1 was strongly expressed in acinar cells as well as in ductal cells. In CP and PCA, VEGF was strongly expressed in ductal cells in CP as well as in cancer cells. We show for the first time that angiogenic activity is increased in both CP and PCA. Based on this study, we suggest that antiangiogenesis might be a novel target for prevention or therapy in chronic pancreatic diseases.     

Comparative studies on the in vitro decidualization process in the baboon (Papio anubis) and human

Blood supply is essential for the maintenance of epididymal function. Since there is no considerable neovascularization in the epididymis, this tissue could represent a suitable model to study the vascular endothelial growth factor (VEGF) effect for vascular permeability. We studied the expression and function of VEGF and its receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase (designated as kinase insert domain-containing receptor, KDR in the human) in the human epididymis. VEGF and VEGF receptors mRNA were detected in the human epididymal tissue. VEGF protein was localized in peritubular and in ciliated cells of efferent ducts as well as in peritubular and basal cells of the epididymal duct. Vascular endothelial cells did not express VEGF. Flt-1 protein was localized in ciliated cells of efferent ducts and in lymphatic vessels. Vascular endothelial cells were negative for Flt-1 but positive for KDR. In vitro VEGF165 treatment of epididymal tissue induced endothelial fenestrations and opening of interendothelial junctions. Additionally, we observed for the first time that VEGF could induce transendothelial gaps. We conclude that these gaps might be of importance not only for molecular transport but also for cell passage across the vessel wall, which may be significant for tumor metastasis. VEGF may act as a paracrine effector to influence the permeability of lymphatic vessels via Flt-1, and of blood vessels via KDR.     

Cellular and subcellular localization of transforming growth factor-alpha and epidermal growth factor receptor in normal and diseased human and hamster pancreas

Four normal pancreas, 8 chronic pancreatitis specimens, and 30 non-endocrine pancreatic tumors from humans and 6 normal and 6 induced pancreatic cancers in hamsters were examined immunohistochemically by antibodies against human transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR). Two normal pancreas and two pancreatic cancer specimens from each species were also studied immunoelectron microscopically by the immunogold method. In chronic pancreatitis, the reactivity and intensity of the staining with both antibodies were much greater in ductal/ductular cells than in the normal pancreas. All 30 pancreatic cancers reacted with both antibodies with a variable degree of reactivity and staining intensity. No correlation was found between the histological type of tumors, the degree of tumor differentiation, and the incidence and patterns of reactivity of either antibody. Immunoelectron microscopically, both EGFR and TGF-alpha were demonstrated primarily on the basal membrane. In the normal hamster pancreas, TGF-alpha was overexpressed in the alpha-cells but not in any other islet cells. Both TGF-alpha and EGFR were marginally detectable in the exocrine pancreas and in induced pancreatic lesions. This is the first demonstration of subcellular localization of TGF-alpha and EGFR in the normal and diseased human and hamster pancreas.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Sociosomatics and illness in chronic fatigue syndrome

Angiogenesis is essential for tumour growth and important in tumour metastasis and prognosis. Vascular endothelial growth factor (VEGF) stimulates endothelial proliferation in vitro and angiogenesis in vivo. VEGF expression has been correlated with high vascularity in tumours, including carcinoma of the breast. This study investigated VEGF expression and vascularity of invasive lobular (n = 10) and invasive ductal carcinoma (n = 28), and pure ductal carcinoma in situ of the breast (n = 33). VEGF protein expression was studied with immunohistochemistry and VEGF mRNA with in situ hybridization. Vascular density was assessed on sections stained for von Willebrand factor. There was more expression of both VEGF protein (P = 0.006) and mRNA (P = 0.002) in invasive ductal than in invasive lobular carcinoma. VEGF protein (rs = 0.32, P = 0.047) and mRNA (rs = 0.56, P = 0.04) correlated with vascular density in invasive ductal carcinoma. In invasive lobular carcinoma, vascular density did not correlate with VEGF mRNA (rs = 0.15, P = 0.35) and was inversely related to VEGF protein (rs = -0.57, P = 0.04). There were no significant differences in vascular density between the two types of invasive carcinoma, suggesting that VEGF is important in angiogenesis in invasive ductal carcinoma, but that other angiogenic factors are important in invasive lobular carcinoma. Although VEGF protein was frequently expressed in ductal carcinoma in situ, no relationship was found between VEGF and the two patterns of angiogenesis previously described.     

Clusterin expression in the early process of pancreas regeneration in the pancreatectomized rat

We have previously reported upregulation of clusterin at the time of islet cell regeneration after beta-cell injury. This led us to speculate that clusterin might be involved in the neogenic regeneration of the pancreas. Clusterin expression was examined throughout the process of pancreatic neogenesis in pancreatectomized rats. For in vitro analysis, duct cells were isolated from the rat pancreas and clusterin cDNA was transfected for its overexpression. Clusterin and its mRNA increased significantly in the early phase of regeneration, particularly at 1-3 days after pancreatectomy. Clusterin was transiently expressed in the differentiating acinar cells but faded afterwards. Interestingly, these clusterin cells were negative for PCNA (proliferating cell nuclear antigen), whereas most epithelial cells in ductules in the regenerating tissue showed extensive proliferative activity. Clusterin expression was also detected in some endocrine cells of the regenerating tissue. Transfection of clusterin cDNA into primary cultured duct cells resulted in a 2.5-fold increase in cell proliferation and induced transformation of non-differentiated duct cells into differentiated cells displaying cytokeratin immunoreactivity. Taken together, these results suggest that clusterin may play essential roles in the neogenic regeneration of pancreatic tissue by stimulating proliferation and differentiation of duct cells.     

Experimental study of transductal dissemination of cancer of the pancreas

Experimental studies on the mechanims of transductal dissemination of the pancreas carcinoma was made in rabbit by injection of VX2 carcinoma suspension into the pancreatic duct. Ductal occlusion was conductive to nidation and growth in the pancreas of intraductal floating cancer cells, but tumor growth also occured in 40% of animals in which the duct was not occluded. The mechanisms of nidation in the pancreas of intraductal floating cancer cells were direct embedding into the ductal wall, and leakage of cancer cells from the duct in the process of pancreatic fibrosis due to ductal obstruction. Expansive tumor growth in the pancreas was more vigorous the smaller the degree of fibrosis of the pancreas, and was most active where associated acute pancreatitis was seen or where the pancreas was X-irradiated one week before injection of carcinoma suspension.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Ovarian function in ewes made hypogonadal with GnRH antagonist and stimulated with FSH in the presence or absence of low amplitude LH pulses

Keratinocyte growth factor (KGF) is an angiogenic and mitogenic polypeptide that has been implicated in cancer growth and tissue development and repair. Its actions are dependent on its binding to a specific cell-surface KGF receptor (KGFR), which is encoded by the fibroblast growth factor (FGF) receptor type II (FGFR-2) gene. In the present study, we compared the immunohistochemical localization of KGF and KGFR/FGFR-2 in the normal and cancerous pancreas using specific antibodies that recognize KGF and KGFR/FGFR-2 and examined the expression of KGF, KGFR, and FGFR-2 in human pancreatic cancer by in situ hybridization with the corresponding riboprobes. In the normal pancreas, KGF immunoreactivity was present principally in the islet cells, whereas KGFR/FGFR-2 immunoreactivity was present both in the islet and ductal cells. In the pancreatic cancers, moderate KGF and moderate to strong KGFR/FGFR-2 immunoreactivity was present in many of the cancer cells. Furthermore, the ductal and acinar cells adjacent to the cancer cells exhibited moderate to strong KGF and KGFR/FGFR-2 immunoreactivity. By in situ hybridization, KGF, KGFR, and FGFR-2 were overexpressed and co-localized in the cancer cells within the pancreatic tumor mass but were even more abundant in the acinar and ductal cells adjacent to the cancer cells. These findings indicate that KGF, KGFR, and FGFR-2 are overexpressed in both the cancer cells and the adjacent pancreatic parenchyma and raise the possibility that KGF may act in an autocrine and paracrine manner to enhance pancreatic cancer cell growth in vivo.     

Factors controlling pancreatic islet neogenesis

We have established a model in which cellophane wrapping induces reiteration of the normal ontogeny of beta-cell differentiation from ductal tissue. The secretion of insulin is physiologic and coordinated to the needs of the animal. Streptozotocin-induced diabetes in hamsters can be 'cured' at least half the time. There appears to be activation of growth factor(s) within the pancreas acting in an autocrine, paracrine or juxtacrine manner to induce ductal cell proliferation and differentiation into functioning beta-cells. Given the results of our studies to date, it does not seem premature to envisage new approaches to the treatment of diabetes mellitus. Identification of the factor(s) which regulates islet cell proliferation and differentiation in our model may permit proto-undifferentiated cells and islets to be grown in culture. This concept could be extended to induce endocrine cell differentiation in vitro as well. Furthermore, islet cell growth factors could be used to provide 'trophic support' to islet transplants as a means of maintaining graft viability. There may also be greater scope for gene therapy when the growth factor(s) has been isolated, purified, sequenced and cloned.     

Acidic calcium pools in intraerythrocytic malaria parasites

AIMS: Angiogenesis is a complex multistep process essential for tumour growth. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and vascular permeability-inducing agent. Recent studies have shown that VEGF expression is correlated to microvessel density and tumour progression. The aim of this study was to analyse VEGF expression in a series of gastrointestinal neuroendocrine tumours. METHODS AND RESULTS: Surgical specimens from 28 gastrointestinal carcinoids and 20 pancreatic endocrine tumours were examined for VEGF expression by immunohistochemistry. Intense cytoplasmic staining for VEGF was observed in several cells of the islets of Langerhans and in neuroendocrine cells of normal digestive mucosa. All midgut carcinoids showed strong VEGF expression in tumoral cells. Positive VEGF immunostaining was observed in 16 of 20 neuroendocrine pancreatic tumours but it was usually much lower than in midgut carcinoids. Western blotting analysis in eight cases identified a major band at 30-32 kDa. No correlation between VEGF expression and tumour stage was found. CONCLUSIONS: This study demonstrates that neuroendocrine cells are a major source of VEGF, particularly in midgut carcinoids. This finding suggests that the presence of VEGF may be required to maintain the differentiated state of capillary vessels in these hypervascular tumours. Such secretion, in conjunction with the other growth factors synthesized by these neuroendocrine tumours, may have an important role in tumour growth.     

Malignant peripheral nerve sheath tumor of bone in children and adolescents

In situ vascular endothelium is characterized by many cytoplasmic vesicles (caveolae) and vacuoles. In venules these are organized into prominent clusters called vesiculo-vacuolar organelles or VVOs. VVOs provide an important pathway for plasma protein extravasation in response to vasoactive mediators. In contrast, cultured endothelial cells isolated from many sources lack VVOs and generally have few caveolae. Our goal was to preserve VVOs in cultured endothelium. Bovine adrenal microvascular endothelial cells (BCEs) cultured on floating Matrigel-collagen Type I gels with vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exhibited typical VVOs by electron microscopy. Both in vivo and in culture VVOs were caveolin-positive by immunoelectron microscopy. On the basis of caveolin immunostaining, VVOs could also be detected by light (confocal) microscopy. When BCEs were cultured without VPF/VEGF, caveolin staining was finely punctate and electron microscopy confirmed the near absence of VVOs. BCE VVOs were sensitive to N-ethylmaleimide. Other types of endothelium cultured on Matrigel-collagen gels with or without VPF/VEGF exhibited few caveolae and no VVOs. Therefore, preservation of VVOs in cultured endothelium required a specific combination of endothelial cells (BCEs), surface matrix (Matrigel-collagen), and growth factor (VPF/VEGF). These endothelial cells should be useful for in vitro studies of trans-endothelial transport.     

Ultrastructural immunogold localization of vimentin and S-100 protein in guinea pig pars tuberalis

All solid tumors must acquire a vascular stroma to grow beyond a minimal size. Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic growth factor both in vitro and in vivo that may participate in the formation of the vascular tumor stroma. In the present study, we examined the expression of VEGF in the paraffin sections of 20 eyes harboring retinoblastoma or posterior uveal melanoma, but also in corresponding tumor cellines. By using in situ hybridization, we found that all but one of the retinoblastomas expressed VEGF mRNA. Particularly high expression was detected in areas of loosely packed tumor cells with prominent chromatin. By contrast, none of the posterior uveal melanomas expressed significant amounts of VEGF mRNA. Immunostaining with an antibody against VEGF confirmed that retinoblastomas, but not posterior uveal melanomas, also contained detectable VEGF protein. To further study the expression of VEGF in these tumor cells we performed Northern blotting on a retinoblastoma celline, Y79, and on an uveal melanoma celline, OM431. Both of these cellines expressed low levels of VEGF mRNA under normal culture conditions. However, when the cells were cultured under hypoxic conditions, a strong increase in VEGF mRNA could be seen in Y79 cells but not in OM431 cells. By using a bioassay, we also found that hypoxia stimulated the secretion of VEGF protein into the culture medium of Y79 cells. In conclusion, we have shown that VEGF mRNA and protein are expressed in retinoblastomas but not in posterior uveal melanomas. Moreover we have shown that VEGF is hypoxia-inducible in retinoblastoma cells. These results suggest that focal hypoxia may act as a stimulus for VEGF production in retinoblastomas, that in turn may contribute to tumor growth by stimulating the formation of a vascular stroma.     

Enhanced expression of vascular endothelial growth factor in metastatic melanoma

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.     

Chronic pancreatitis is associated with increased concentrations of epidermal growth factor receptor, transforming growth factor alpha, and phospholipase C gamma

The epidermal growth factor (EGF) receptor is a transmembrane protein that binds EGF and transforming growth factor alpha (TGF alpha), and that stimulates phospholipase C gamma 1 (PLC gamma 1) activity. In this study the role of the EGF receptor in chronic pancreatitis was studied. By immunohistochemistry, the EGF receptor, TGF alpha, and PLC gamma 1 were found to be expressed at high concentrations in pancreatic ductal and acinar cells from chronic pancreatitis patients. Northern blot analysis showed that, by comparison with normal controls, 19 of 27 chronic pancreatitis tissues exhibited a 5.7-fold increase in EGF receptor mRNA concentrations, and 20 of 27 chronic pancreatitis tissues exhibited a sixfold increase in TGF alpha mRNA concentrations. In situ hybridisation confirmed that overexpression occurred in ductal and acinar cells, and showed that both mRNA moieties colocalised with their respective proteins. These findings suggest that TGF alpha may act through autocrine and paracrine mechanisms to excessively activate the overexpressed EGF receptor in the two major cell types of the exocrine pancreas, thereby contributing to the pathobiology of this disorder.     

In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-1, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

The effect of linomide on the migration and the proliferation of capillary endothelial cells elicited by vascular endothelial growth factor

In order to assess the mechanism of action of the quinoline-3-carboxyamide linomide as an antiangiogenic drug, the effect of linomide was studied in vitro on postcapillary endothelial cells exposed to vascular endothelial growth factor (VEGF). Linomide did not block the spontaneous replication of endothelial cells, but significantly suppressed endothelial cell growth and migration elicited by VEGF. It is concluded that linomide appears to be an effective tool to inhibit VEGF-dependent angiogenesis.