Endothelial Cells Of Microvessels Research Articles

Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells.

Endothelial regeneration and angiogenesis in the intima of the arterial wall are key events in the pathogenesis of transplantation arteriosclerosis. The traditional hypothesis that damaged endothelial cells are replaced by remaining cells of the donor vessel has been challenged by recent observations, but the cell origins of large arteries and microvessels are still not well established. Aortic segments were allografted between Balb/c and TIE2-LacZ (C57BL/6) mice expressing beta-galactosidase (gal) in endothelial cells. beta-gal+ cells in TIE2-LacZ vessels grafted to Balb/c mice completely disappeared, whereas the positive cells found in Balb/c aorta allografted into TIE2-LacZ mice 4 weeks after surgery indicated a host origin. En face analysis of allograft vessels displayed a unique distribution of beta-gal+ cells on the surface at 3 days, 1 week, and 4 weeks. Interestingly, 35+/-19% beta-gal+ cells were found in arterial segments allografted into chimeric mice with TIE2-LacZ bone marrows. Furthermore, endothelial cells of microvessels within allografts had a beta-gal+ staining in the media at 1 week and in the neointimal lesions and adventitia at 4 weeks. Allograft studies in chimeric mice demonstrated that beta-gal+ cells of microvessels in transplant arteriosclerosis were derived from bone marrow progenitors. We provide strong evidence that endothelial cells of neointimal lesions in allografts are derived from circulating progenitor cells and that bone marrow-derived progenitors are responsible for angiogenesis of the allograft, that is, the formation of microvessels in transplant arteriosclerosis.

Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules.

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-alpha, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.

The involvement of vascular endothelial growth factor and flt-1 in the process of neointimal proliferation in pig coronary arteries following stent implantation.

To clarify the role of vascular endothelial growth factor (VEGF) in the process of restenosis, a Palmaz-Schatz stent was implanted in the left anterior descending coronary artery of male pigs at 2 weeks after balloon injury (balloon/artery ratio 1.2:1). The animals were euthanized at 1, 2, and 4 weeks after stenting, and western blot and immunohistochemical analysis were performed using VEGF, fms-like tyrosine kinase (flt)-1, and platelet-derived growth factor (PDGF) antibodies. The expressions of VEGF and flt-1 protein in the neointima were observed as early as 1 week after stenting and remained for up to 4 weeks, while re-endothelialization was complete at 2 weeks. These protein expressions were demonstrated in proliferated smooth muscle cells throughout the entire period after stenting and, in addition, they were observed in the macrophages and endothelial cells of microvessels around stent struts at 4 weeks. The expression pattern of VEGF corresponded with that of PDGF, a growth factor well-known to induce neointimal proliferation. The cell proliferative activity, measured by the proliferating cell nuclear antigen index, around the struts remained high until 4 weeks after stenting, while that in the other areas declined at 4 weeks. These results suggest that VEGF is involved in the process of restenosis not only through its angiogenic properties and induction of monocyte chemotaxis, but also by a synergistic effect with PDGF.

Increased Binding Activity at an Antioxidant-Responsive Element in the Metallothionein-1 Promoter and Rapid Induction of Metallothionein-1 and -2 in Response to Cerebral Ischemia and Reperfusion

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are potentially involved in zinc homeostasis and free radical scavenging. The expression pattern of MT-1 and the binding activity of various MT-1 promoter elements were investigated after mild focal cerebral ischemia in the rat. Transient focal ischemia was induced by occluding both common carotid arteries and the right middle cerebral artery for 30 min. By the use of real-time quantitative PCR, a 10-fold increase in MT-1 and -2 mRNA levels was found in the cortex 24 hr after reperfusion. In situ hybridization and immunocytochemistry showed a rapid increase in MT-1 and -2 mRNA and MT protein in endothelial cells of microvessels at 6 hr after reperfusion, followed by an increased expression in astrocytes of the infarcted cortex at 24 hr after reperfusion. The early increase in MT expression preceded an increase in cerebral edema measured with T2-weighted magnetic resonance imaging. Gel shift assays were performed on nuclear extracts prepared from cortices before and at 6 and 24 hr after reperfusion. Increased binding activity was found at an antioxidant/electrophilic response element (ARE) sequence in the MT-1 promoter at 6 hr with a lower and variable binding activity at 24 hr after reperfusion. Constitutive binding activity was found for Sp1 and a metal response element in the MT-1 promoter that did not increase after ischemia and reperfusion. This study suggests a role of ARE-binding proteins in inducing cerebral MT-1 expression and implicates MT-1 as one of the early detoxifying genes in an endogenous defense response to cerebral ischemia and reperfusion.

Angiogenesis in cutaneous lymphoproliferative disorders: microvessel density discriminates between cutaneous B-cell lymphomas and B-cell pseudolymphomas.

The role of angiogenesis in neoplastic disorders is supported by the evidence that tumor growth beyond a certain size requires induction of new blood vessels. The extent of tumor-associated angiogenesis, measured as microvessel density (MVD), has shown to correlate with aggressiveness and the prognostic outcome in several malignant neoplasms. Few data have been reported on the angiogenic response in lymphoproliferative diseases. In this study, the MVD has been assessed in benign and malignant primary cutaneous B-cell lymphoproliferative disorders. MVD was determined in formaldehyde-fixed, paraffin-embedded specimens of primary cutaneous B-cell lymphomas (CBCL; n = 18) and cutaneous B-cell pseudolymphomas (B-PSL; n = 22) according to previously described protocols but was performed using computer-aided microscopic morphometry. The endothelial cells of microvessels were identified by immunohistochemical staining for factor VIII-related antigen and CD31. The MVD was 99 dots/mm2 for CBCL and 68 dots/mm2 for PSL, and a MVD of 115 dots/mm2 for CBCL and 73 dots/mm2 for PSL by using an antibody against factor VIII-related antigen and CD31 antigen, respectively. Univariate analysis revealed statistically highly significant differences in MVD between CBCL and B-PSL (P = 0.0036 with staining for factor VIII-related antigen and P = 0.0002 with staining for CD31 antigen). This study analyzes for the first time the angiogenic response in CBCL compared with that of B-PSL and demonstrates that MVD discriminates between CBCL and B-PSL. However, because of an overlap in the ranges of MVD in CBCL and PSL, the MVD is not useful as a diagnostic tool in individual cases.

Increased in situ expression of nitric oxide synthase in human colorectal cancer.

There is growing evidence that nitric oxide (NO) has an important role in tumor growth. However, information on the expression of NO synthase (NOS) in colorectal cancers is scanty. We therefore investigated the distribution and expression of NOS in human colorectal cancers. The expression of three types of NOS, inducible (iNOS), endothelial (eNOS) and neuronal (nNOS), was examined by immunohistochemistry in 25 cases of colorectal cancer. The expression of iNOS was also investigated at the mRNA level using the reverse transcriptase polymerase chain reaction (RT-PCR) in 6 cases. Correlations were made between iNOS expression and the histopathological findings. Immunoreactive iNOS was detected in the tumor cells in 22 cases (88%) with diffuse cytoplasmic reactions. Expression of iNOS-mRNA detected by RT-PCR in three tumor tissues was over five-fold that in normal mucosa. Intensified immunoreactivity of iNOS was associated with vascular invasion. iNOS expression did not correlate with pathological staging, tumor size, lymph node metastasis, p53 expression or tumor vessel density. Immunoreactive eNOS stained more strongly in the endothelial cells of microvessels within and around the tumor than in the areas remote from the tumor. There is enhanced expression of iNOS and eNOS in human colorectal cancers, which may correlate with tumor growth and vascular invasion.

Possible reverse transport of beta-amyloid peptide across the blood-brain barrier.

Our experiments were performed to test the hypothesis that human beta-amyloid peptide 42 (beta A) is able to enter and exit the brain parenchyma through the blood-brain barrier. In an effort to determine the effect of beta A in an animal model, we have injected beta A i.v. into rats following single and repeated brain ischemia. Rats were sacrificed at 3 and 12 months after injection and beta A was localized by monoclonal antibody (mAb) 4G8. The present observations revealed an abundant presence of beta A in the extracellular space of the brain, which appeared to be dilated, and a vigorous uptake of beta A into the cytoplasm of endothelial and ependymal cells, pericytes, astrocytes and neurons. Some of the beta A deposits were associated and/or had migrated to the vessels and to the ventricles, and by 3 months a significant amount of beta A was directly associated with the vessels and was observed inside the ventricular space. Virtually no soluble and aggregating beta A was found in brain tissue 1 year later. This suggests that phagocytic pericytes and astrocytes take up exogenous beta A in an attempt to clear the peptide from the brain extracellular space and deliver it to the circulation. Further, direct removal of beta A from the ventricles by the bloodstream is also possible. These observations suggest that a reverse transport of beta A across endothelial cells of microvessels represents one of the possible mechanisms responsible for removal of extravasated beta A. The findings of the present study indicate that in normal conditions beta A is rapidly cleared from the cerebrospinal fluid and brain parenchyma, suggesting that irreversible changes in the physico-chemical properties of the cerebrovascular endothelial cell surface are involved in beta A deposition in the brain in Alzheimer's disease (AD).

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells.

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-gamma, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-gamma-inducible CXC chemokines--IFN-inducible protein 10 (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha chemoattractant (I-TAC)--by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MO) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MO, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1beta, TNF-alpha, and CD40 ligand potentiated IP-10 expression from IFN-gamma-stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-gamma induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis.

Microvascular permeability.

This review addresses classical questions concerning microvascular permeabiltiy in the light of recent experimental work on intact microvascular beds, single perfused microvessels, and endothelial cell cultures. Analyses, based on ultrastructural data from serial sections of the clefts between the endothelial cells of microvessels with continuous walls, conform to the hypothesis that different permeabilities to water and small hydrophilic solutes in microvessels of different tissues can be accounted for by tortuous three-dimensional pathways that pass through breaks in the junctional strands. A fiber matrix ultrafilter at the luminal entrance to the clefts is essential if microvascular walls are to retain their low permeability to macromolecules. Quantitative estimates of exchange through the channels in the endothelial cell membranes suggest that these contribute little to the permeability of most but not all microvessels. The arguments against the convective transport of macromolecules through porous pathways and for the passage of macromolecules by transcytosis via mechanisms linked to the integrity of endothelial vesicles are evaluated. Finally, intracellular signaling mechanisms implicated in transient increases in venular microvessel permeability such as occur in acute inflammation are reviewed in relation to studies of the molecular mechanisms involved in signal transduction in cultured endothelial cells.

Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions: possible pathophysiological significance of VEGF in progression of atherosclerosis.

Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription-polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression. Hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Matrix metal loproteinases-2 and -9, and tissue inhibitor of metal loproteinases-1 of the sheep placenta during the last third of gestation

Uteroplacental and fetoplacental blood flows increase dramatically during the last third of gestation. Therefore, angiogenesis and tissue remodelling may regulate placental growth. On days 110, 120, 130, and 142 (term = 145 ± 3; n = 5 ewes/day) of gestation, sheep placentae were separated into maternal (caruncular and intercaruncular) and fetal (cotyledonary and intercotyledonary) components; tissues were minced and cultured (24 h) and media were analysed for gelatinase activity and for tissue inhibitor of metalloproteinases-1 (TIMP-1) protein. Both the 72-kDa (matrix metalloproteinase-2; MMP-2) and 95-kDa (matrix metalloproteinase-9; MMP-9) gelatinases were detected by zymography. There was more ( P<0.001) MMP-2 activity in the caruncle versus intercaruncle and intercotyledon, and all had greater MMP-2 activity ( P<0.001) than the cotyledon. There was less ( P<0.04) MMP-9 in the intercotyledon than in the other components. By Western analysis, there was more ( P<0.004) TIMP-1 (30 kDa) in the caruncle-conditioned media compared to the other tissues, which did not differ from each other ( P≥0.38). The immunohistochemical staining for MMP-2, MMP-9, and TIMP-1 determined that staining was associated with the epithelial cells of the intercotyledon, the uterine glands, cells of the fetomaternal interface of the placentome (trophoblast and binucleate cells), and the endothelial cells of microvessels. Therefore, MMP-2, MMP-9, and TIMP-1 are present in the last third of gestation, suggesting that there is an ongoing tight regulation of tissue remodelling and growth throughout this critical time of pregnancy.

First-pass metabolism of peptide drugs in rat perfused liver.

To elucidate the extent and mechanisms of the first-pass metabolism of peptide drugs in the liver after oral administration, a liver perfusion study was performed in rats using metkephamid, a stable analogue of methionine enkephalin, and thyrotropin-releasing hormone (TRH), as model peptides. The fraction of intact metkephamid recovered after single-pass constant perfusion through rat liver reached steady-state very quickly, and it was concluded that metkephamid was hydrolysed enzymatically at the surface of hepatocytes or endothelial cells of microvessels, or both, rather than being taken up by hepatocytes. The fraction of metkephamid recovered intact was approximately 40% under protein-free conditions but increased to 70-75% on addition of bovine serum albumin (BSA) to the perfusate. The fraction of metkephamid bound to BSA was approximately 50% under these conditions, implying that only the free fraction of metkephamid in the plasma was metabolized in the liver. Calculations based on the tube model showed that approximately 30-35% of metkephamid absorbed from the intestine undergoes first-pass metabolism before entering the systemic circulation in-vivo. In contrast, the fraction of TRH metabolized in the liver was less than 10%, indicating a remarkably low contribution of first-pass metabolism to the bioavailability of TRH. These results show that hepatic first-pass metabolism of metkephamid contributes to its low systemic bioavailability. After intestinal absorption free metkephamid is rapidly hydrolysed on the surface of hepatocytes or endothelial cells, rather than being taken up by hepatocytes. This information has important implications in the oral delivery of many kinds of peptide.

Expression of fibroblast growth factor-2 and fibroblast growth factor receptor-1 in thyroid diseases: difference between neoplasms and hyperplastic lesions.

We studied the expression of both fibroblast growth factor-2 (FGF-2) and FGF receptor-1 (FGFR-1) in various histological types of human thyroid neoplastic and hyperplastic tissues to clarify the biological behavior of FGF-2. A total of 37 malignant tumors (24 papillary carcinomas, 10 follicular carcinomas, 3 anaplastic carcinomas), 8 follicular adenomas, and 12 adenomatous goiters were examined by immunohistochemical methods. With immunohistochemical staining, both FGF-2 and FGFR-1 were frequently detected in human thyroid carcinoma (79.2 to 100% and 80 to 100%, respectively). In thyroid hyperplastic lesions such as adenomatous goiter, the FGF-2 immunoreactivity in follicular cells was detected in 2 of 12 adenomatous goiters (16.7%). In contrast, FGFR-1 immunoreactivity was detected in 66.7% of cases of this disease. The endothelial cells of microvessels in the stroma adjacent to the neoplasms and hyperplastic lesions also showed cytoplasmic FGF-2 immunoreactivity. The difference between FGF-2 and FGFR-1 expression in adenomatous goiters was statistically significant (P<0.05). Furthermore, the difference in FGF-2 immunoreactivity between carcinoma and adenomatous goiter was statistically significant (P=0.0001). The present investigation demonstrated the possibility of an autocrine mechanism of action of FGF-2 in human thyroid carcinoma. Moreover, in thyroid hyperplastic lesions, FGF-2 derived from the stroma might be involved in the formation of nodular and/or diffuse goiters.

Tumor angiogenesis predicts recurrence in invasive colorectal cancer when controlled for Dukes staging.

Tumor angiogenesis is associated with metastasis in several types of solid tumors, including melanoma, breast, prostate, lung, bladder, and oral-cavity tumors. The purpose of this study was to determine whether tumor angiogenesis could predict recurrence following curative surgery for colorectal cancer. Thirty-five patients were studied, including 13 patients with recurrent tumor and 22 without. Representative formalin-fixed, paraffin-embedded sections of invasive colorectal cancers from these patients were sectioned. The endothelial cells of microvessels within the tumors were highlighted by immunohistochemical staining for CD31. The most active areas were identified and the microvessels counted in a x 400 field (0.152 mm2) by two observers in a blinded fashion. Tumor microvessel count (p = 0.0062). Dukes' staging (p = 0.0004), vascular invasion (p = 0.0280), and tumor grade (p = 0.0559) were all significantly associated with tumor recurrence. Tumor microvessel counts > or = 65 per x 400 field were associated with tumor recurrence (p = 0.0035, relative risk [RR] = 11.3). Controlling for Dukes' stage, a multivariate logistic regression model revealed that a tumor microvessel count > or = 65 is an important predictor of tumor recurrence (p = 0.0783, RR = 6.0). A backwards elimination proportional hazards model revealed that a microvessel count > or = 65 shows a trend toward independent prediction of time to tumor recurrence (p = 0.1203, RR = 2.967) when controlled for Dukes' staging (p = 0.0029, RR = 9.089). Despite the small number of patients studied, these results suggest that the number of microvessels in sections of invasive colorectal adenocarcinoma immunohistochemically stained with CD31 may be an important independent predictor of tumor recurrence and time to recurrence.

Role of Leukotriene-degrading enzymes in pulmonary response to antigen infusion in sensitized guinea pigs in vivo.

To determine the role of leukotriene (LT)-degrading enzymes in allergic reactions, we studied the effects of inhibitors of gamma-glutamyl transpeptidase (gamma-GTP) and dipeptidases on increases in pulmonary insufflation pressure (PIP) and vascular permeability induced by ovalbumin (OA) antigen in guinea pigs sensitized to OA antigen in vivo. Vascular permeability was assessed by the amount of extravasated Evans blue dye from the trachea, main bronchi, and segmental bronchi. An intravenous (i.v.) administration of OA antigen (200 micrograms/kg) caused increases in PIP and extravasated Evans blue dye, and OA antigen-induced effects were potentiated by gamma-GTP inhibitor L-serine borate (3 x 10(-5) M/kg, i.v.) (P < 0.05) and an inhibitor of dipeptidases, L-cysteine (3 x 10(-5) M/kg, i.v.) (P < 0.01). OA antigen-induced increases in PIP and Evans blue dye extravasation were in part inhibited by LT-receptor antagonist ONO-1078 (10(-4) M/kg, i.v.). Guinea-pig tracheal tissues contained gamma-GTP and microsomal dipeptidase activities. Histochemical and immunohistochemical studies indicate that gamma-GTP-like activity existed in the epithelium and smooth muscle, and an activity of microsomal dipeptidase was observed in the endothelial cells of microvessels and epithelium. These results suggest that LT-degrading enzymes have an important role in regulating allergic reaction in the airway in vivo.

Endothelin peptides in brain diseases.

Recently, scientists interested in diseases of the human brain have paid much attention to the endothelin group of peptides. Under normal conditions they are found in some types of neurons and in endothelial cells of microvessels but not in glial cells. This review focuses on the endothelin peptides and their involvement in various brain diseases. Particular attention is paid to their expression in reactive astrocytes seen in many pathological conditions of the human brain. Endothelin-1 is a very potent vasoconstrictor which may be involved in the vasospasm occurring in subarachnoid haemorrhage. Intracerebral injection or application to cerebral arteries in animals will cause a focal necrosis, apparently due to severe vasoconstriction. Reactive astrocytes occurring in cases with infarcts, lacunae, Alzheimer's disease, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE) express endothelin-like immunoreactivity. Astrocytes in vitro may produce, store and release endothelins. To some extent astrocytes grown in vitro mimic reactive astrocytes in vivo since in cultures astrocytes are removed from their natural environment which may trigger reactive responses. Therefore, in vivo reactive astrocytes may produce, store and release endothelins just as in vitro. If endothelins are released from reactive astrocytes they may act as mitogens and may influence microcirculation by inducing vasoconstriction of intracerebral arterioles. In such ways endothelins may contribute to the final lesions seen in cases with infarcts, lacunae, traumatic conditions, Alzheimer's disease and inflammatory diseases of the brain.

Cathepsin B in angiogenesis of human prostate: An immunohistochemical and immunoelectron microscopic analysis

Angiogenesis (or neovascularization) is required for the growth of solid organ tumors and precedes invasion of the adjacent stroma by neoplastic cells. We investigated the relative density and distribution of cathepsin B (CB) immunostained microvessels (i.e., small blood vessels and capillaries) in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma (CAP) by immunocytochemical localization of an antibody directed against a cathepsin B-derived synthetic peptide (Syn-CB). We studied 16 formalin-fixed, prostatectomy specimens that were embedded in paraffin/paraplast for histological examination by hematoxylin and eosin and immuno-localization of the Syn-CB antibody. Selected paraformaldehyde-fixed specimens were embedded in K4M Lowicryl or LRWhite resins. We localized the antibody in thin sections using immunoelectron microscopy techniques. Eight patients had BPH [4 patients with BPH alone, 2 with BPH and PIN, and 2 with BPH and CAP]. Ten cancer cases included one with Gleason histologic score 4, two with score 6, four with score 7, and three with score 8. In CAP cases, Gleason score 6 and 7 tumors had more microvessels than the score 4 or 8 tumors. In both BPH and CAP cases, the antibody was localized chiefly in the endothelial cells of microvessels, but occasionally in ductal and glandular epithelial cells. Ultrastructurally, CB-immunoreactive gold particles were markedly increased at the luminal and basal plasma membrane surfaces and folds of endothelial cells in neoplastic prostate, but not in the endothelial cells of BPH. Furthermore, the presence of CB localizing gold particles in collagen and smooth muscle fibers near the microvessels indicated leakage of the enzyme in prostatic stroma of neoplastic prostate. Similar leakage was not observed in BPH. Morphometric analysis showed that the relative density of microvessels increased two to three times in cancer patients when compared to patients with BPH alone. Our study also indicated that BPH associated with PIN or CAP had an increased density of microvessels when compared to BPH alone. Our study showed that the relative density and distribution of microvessels are the most important features of neovascularization in prostatic tumors. The relative density of microvessels increased in PIN and CAP when compared to BPH alone. Although the localization of CB is associated with lysosomes of endothelial cells in both BPH and CAP, there is a greater association of CB with the plasma membranes of endothelial cells in CAP than BPH. Immunoelectron microscopy provided evidence that CB might be involved in dissolution of basement membranes in neoplastic tumors during angiogenesis. CB localization has the potential of defining a role for this protease in degradation of extracellular matrix constituents during early steps of angiogenesis.

Vascular tree of human placenta in complicated pregnancy

Uteroplacental and fetoplacental blood flows increase dramatically during the last third of gestation. Therefore, angiogenesis and tissue remodelling may regulate placental growth. On days 110, 120, 130, and 142 (term = 145 ± 3; n = 5 ewes/day) of gestation, sheep placentae were separated into maternal (caruncular and intercaruncular) and fetal (cotyledonary and intercotyledonary) components; tissues were minced and cultured (24 h) and media were analysed for gelatinase activity and for tissue inhibitor of metalloproteinases-1 (TIMP-1) protein. Both the 72-kDa (matrix metalloproteinase-2; MMP-2) and 95-kDa (matrix metalloproteinase-9; MMP-9) gelatinases were detected by zymography. There was more (P<0.001) MMP-2 activity in the caruncle versus intercaruncle and intercotyledon, and all had greater MMP-2 activity (P<0.001) than the cotyledon. There was less (P<0.04) MMP-9 in the intercotyledon than in the other components. By Western analysis, there was more (P<0.004) TIMP-1 (30 kDa) in the caruncle-conditioned media compared to the other tissues, which did not differ from each other (P≥0.38). The immunohistochemical staining for MMP-2, MMP-9, and TIMP-1 determined that staining was associated with the epithelial cells of the intercotyledon, the uterine glands, cells of the fetomaternal interface of the placentome (trophoblast and binucleate cells), and the endothelial cells of microvessels. Therefore, MMP-2, MMP-9, and TIMP-1 are present in the last third of gestation, suggesting that there is an ongoing tight regulation of tissue remodelling and growth throughout this critical time of pregnancy.

Age-related changes in the microvessels of the human stomach: an ultrastructural study.

The ultrastructure of microvessels in the gastric mucosa of patients with atrophic gastritis was investigated. In the gastric mucosa of atrophic gastritis, the endothelial cells of microvessels were irregularly swollen and subendothelial spaces were enlarged. The basement membrane underlying the endothelium was thickened. Degeneration of smooth-muscle cells was also evident in the media. These changes appear to correspond to sclerosis or aging of the microvessels. Such sclerotic changes in microvessels may initiate deterioration of the microenvironment in the gastric mucosa and may consequently affect metabolic activity of the gastric mucosal cells. Those sequences might induce degeneration or abnormal differentiation of mucosal cells. Such vascular changes may advance slowly and increase in intensity with advancing age. The final result would be the histologic changes associated with atrophic gastritis.

Action of tumor necrosis factor on the microvascular endothelium and its role in morphological changes in the internal organs

Tumor necrosis factor exerts the systemic influence on endothelial cells in vivo. Structural changes in endotheliocytes lead to specific damage in the organs. Interstitial and alveolar edema develops in lung. Kupffer cell activation, change of sinusoid endothelial cell porosity, lipid dystrophia of hepatocytes are revealed in liver, damage of glomerular and peritubular capillaries, dystrophic changes of tubular epithelial cells are found out in kidney. The data obtained indicate that endothelial cells in microvessels are one of major cellular targets for action of tumor necrosis factor.