The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

An autocrine loop mediates expression of vascular endothelial growth factor in human dermal microvascular endothelial cells.

The expression of vascular endothelial growth factor mRNA and protein is regulated by a number of agents including growth factors, cytokines, and phorbol esters. Here we report that vascular endothelial growth factor is able to increase its own level in cultured human dermal microvascular endothelial cells. Accumulation of vascular endothelial growth factor mRNA and polypeptide can be detected as early as 4 h after addition of vascular endothelial growth factor to the cell culture medium. The autocrine action of vascular endothelial growth factor appears to be mediated by the KDR receptor. The increase of its own message by vascular endothelial growth factor is blocked by the transcription inhibitor actinomycin D. Transient transfection experiments performed with human dermal microvascular endothelial cells and using a 3.2 kb human vascular endothelial growth factor promoter fragment showed that vascular endothelial growth factor auto-induction can be mimicked at the promoter level. This indicates that the observed vascular endothelial growth factor mRNA increase after vascular endothelial growth factor treatment is occurring at the level of transcription. Furthermore, vascular endothelial growth factor auto-induction is inhibited by PD 098059, showing that phosphorylation events, catalyzed by mitogen activated protein kinases, are a prerequisite for the vascular endothelial growth factor effect. Examination of extracellular signal-regulated kinase and c-Jun N-terminal protein kinase catalytic activities showed that both enzymes have to be activated to mediate the vascular endothelial growth factor signal. Our data demonstrate for the first time the existence of an autocrine loop for vascular endothelial growth factor in endothelial cells. Most probably this represents an amplification mechanism for the action of vascular endothelial growth factor in the microvascularization process.

Telomerized human microvasculature is functional in vivo.

Previously we showed the superior in vitro survival of human telomerase reverse transcriptase (hTERT)-transduced human endothelial cells (EC). Here we show that retroviral-mediated transduction of hTERT in human dermal microvascular EC (HDMEC) results in cell lines that form microvascular structures when subcutaneously implanted in severe combined immunodeficiency (SCID) mice. Anti-human type IV collagen basement membrane immunoreactivity and visualization of enhanced green fluorescent protein (eGFP)-labeled microvessels confirmed the human origin of these capillaries. No human vasculature was observed after implantation of HT1080 fibrosarcoma cells, 293 human embryonic kidney cells, or human skin fibroblasts. Intravascular red fluorescent microspheres injected into host circulation were found within green "telomerized" microvessels, indicating functional murine-human vessel anastamoses. Whereas primary HDMEC-derived vessel density decreased with time, telomerized HDMEC maintained durable vessels six weeks after xenografting. Modulation of implant vessel density by exposure to different angiogenic and angiostatic factors demonstrated the utility of this system for the study of human microvascular remodeling in vivo.

Differential expression of nitric oxide by dermal microvascular endothelial cells from patients with scleroderma.

Vascular abnormalities in scleroderma are fundamental to the pathogenesis of this disease. The objective of this study was to characterize dermal microvascular endothelial cells (DMEC) isolated from scleroderma patients with respect to growth and expression of the constitutive form of endothelial nitric oxide synthase (eNOS). DMEC from patients with both systemic sclerosis (SSc) and localized scleroderma (Loc Scl) contained small intact microvascular structures in contrast to single cell isolations obtained from control skin. Immunoaffinity selection on anti-PECAM-1 beads yielded pure populations of DMEC expressing normal markers. While the morphology and initial growth of SSc DMEC closely paralleled control cells, the growth of SSc DMEC decreased with time in culture (doubling time of 3 days vs. 5 days). Expression of ecNOS mRNA was reduced in both Loc Scl and SSc as shown by semi-quantitative RT-PCR (p < 0.001). Western blots showed variable but generally lower ecNOS protein levels and decreased levels of nitrogen oxides in media were found from both SSc and Loc Scl relative to control cells. The results indicate an intrinsic defect in the mechanism of nitric oxide production in DMEC isolated from scleroderma patients and suggest its possible involvement in the pathophysiology of scleroderma.

Human endothelial cell life extension by telomerase expression.

Normal human endothelial cells, like other somatic cells in culture, divide a limited number of times before entering a nondividing state called replicative senescence. Expression of the catalytic component of human telomerase, human telomerase reverse transcriptase (hTERT), extends the life span of human fibroblasts and retinal pigment epithelial cells beyond senescence without causing neoplastic transformation (Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998) Science 279, 349-352; Jiang, X., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A., Wahl, G., Tlsty, T., and Chiu, C.-P. (1999) Nat. Genet. 21, 111-114). Here, we show that both human large vessel and microvascular endothelial cells also bypass replicative senescence after introduction of hTERT. For the first time, we report that hTERT expression in these life-extended vascular cells does not affect their differentiated and functional phenotype and that these cells maintain their angiogenic potential in vitro. Furthermore, hTERT(+) microvascular endothelial cells have normal karyotype, and hTERT(+) endothelial cell strains do not exhibit a transformed phenotype. Relative to parental cells at senescence, hTERT-expressing endothelial cells exhibit resistance to induction of apoptosis by a variety of different conditions. Such characteristics are highly desirable for designing vascular transplantation and gene therapy delivery systems in vivo.

Compound heterozygosity for novel splice site mutations in the BPAG2/COL17A1 gene underlies generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa, GABEB (OMIM# 226650), is a nonlethal variant of epidermolysis bullosa with autosomal recessive inheritance pattern. The pathogenesis of this disorder can be caused by mutations affecting two different gene/protein systems. Most of the mutations have been identified in the BPAG2/COL17A1 gene encoding a hemidesmosomal transmembrane protein, the 180 kDa bullous pemphigoid antigen (BP180), also known as type XVII collagen. The minority of the mutations are localized in the LAMB3 gene encoding the beta3 polypeptide of laminin 5. In In this study we describe a GABEB patient who showed absent expression of BP180 in the cultured keratinocytes as well as in the skin. The patient was a compound heterozygote for two different splice site mutations, 3053-1G-->C and 3871+1G-->C, affecting the extra-cellular domain of the protein. These mutations resulted in multiple aberrant splice variants, three of them causing premature termination codons for translation. This case, dealing with out-of-frame splice site mutations in BPAG2/COL17A1, attests to the molecular heterogeneity of GABEB.

Membrane-type matrix metalloproteinases in human dermal microvascular endothelial cells: expression and morphogenetic correlation.

Membrane-type matrix metalloproteinases (MT-MMP) activate the zymogen form of MMP-2/Gelatinase A on cell surfaces and are expressed in invasive tumors. We sought to identify and characterize MT-MMP in a non-malignant cell type that undergoes a physiologic and reversible invasive phenotype during angiogenesis. Human dermal microvascular endothelial cells (HDMEC) were isolated from neonatal tissue and purified by anti-CD31 (PECAM) affinity beads. MT-MMP-1 and -3 transcripts were amplified by reverse transcriptase-polymerase chain reaction and northern blots showed a single 4.5 kB mRNA for MT-MMP-1 that was modulated by angiogenic factors and phorbol ester. Immunoblotting of reduced cellular extracts with different MT-MMP-1 antibodies showed the presence of the 63-65 kDa and 57-60 kDa forms, as well as additional forms at lower molecular weights. HDMEC membranes extracted with Triton X114 were incubated with gelatin-sepharose purified MMP-2 and MMP-9 to show activation of proenzymes. Pre-incubation of HDMEC with anti-MT-MMP-1 antibodies decreased proMMP-2 conversion activity only. The movement of HDMEC and the formation of tubule-like structures in three-dimensional collagen gels was markedly delayed by preincubation with the same anti-MT-MMP-1 antibodies. These results demonstrate the presence of MT-MMP in cutaneous microvascular cells in vitro. Modulation of these cell surface proteinases by angiogenic factors, demonstration of multiple processed forms, and specific attenuation of HDMEC morphogenetic patterns in three-dimensional collagen gels implicate their potential roles in the formation of new blood vessels in the skin.

Vascular abnormalities in scleroderma.

Vascular abnormalities represent a fundamental event in the pathogenesis of systemic sclerosis. This review focuses on key observations that support this view and builds a framework with which to clarify our understanding of how endothelial cell damage may trigger a self-fueling process ending in pathological tissue fibrosis in those susceptible to scleroderma. The studies reviewed in this article pertain to systemic sclerosis patients.

Interleukin-1 induces major phenotypic changes in human skin microvascular endothelial cells.

To determine the role of the pleiotropic cytokine interleukin-1 (IL-1) on the activation of endothelial cells during inflammation and angiogenesis, pure populations of human dermal microvascular endothelial cells (HDMEC) were obtained by immunoaffinity purification using Ulex europaeus agglutinin-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody. Exposure of HDMEC to IL-1beta induced morphologic and physiologic changes characterized by 1) phenotypic modulation of endothelial cells from epithelioid to spindle-shaped cells accompanied by reorganization of vimentin filaments; 2) gradual decrease to a complete absence of the endothelial cell markers von Willebrand factor (vWf) and PECAM-1; and 3) increased capability to form tubule-like structures when overlaid with collagen gels. The IL-1 effect on cell morphology, growth, and decrease of endothelial cell antigens was potentiated by basic fibroblast growth factor (bFGF). Similar results were observed in mitotically arrested gamma-irradiated cells demonstrating that the spindle-shaped cells observed after IL-1 stimulation were derived from epithelioid endothelial cells and that DNA synthesis was not required to effect these changes. Immunostaining with an antibody specific for human fibroblasts was negative and further confirmed the endothelial cell origin of the spindle-shaped cells. These data demonstrate that IL-1 can induce phenotypic changes in HDMEC from epithelioid to spindle-shaped, mesenchymal-like cells, that these cells are more susceptible to stimulation by bFGF, and that they lose biochemical and functional properties characteristic of epithelioid HDMEC.

LAD-1 is absent in a subset of junctional epidermolysis bullosa patients.

The anchoring filament protein LAD-1 has been recently identified as the target of autoantibodies in the acquired blistering disorder linear IgA bullous dermatosis. Because this protein appears to be involved in the process of dermal-epidermal cohesion, this study sought to determine the involvement of LAD-1 in the pathology of junctional epidermolysis bullosa (JEB). To this end, 44 patients with a variety of subtypes of JEB were analyzed by indirect immunofluorescence microscopy with antibodies to LAD-1, BP180, and laminin-5. We found that only patients with generalized atrophic benign epidermolysis bullosa (GABEB) contained LAD-1 defects. Of the 16 GABEB patients studied, 13 showed absent or greatly reduced expression of LAD-1 (including 2 patients with a peculiar interrupted staining pattern) and 3 patients showed defects of laminin-5 expression with normal LAD-1 expression. Patients who showed LAD-1 defects also showed abnormal expression of BP180. Keratinocytes were cultured from the skin of two GABEB patients and analyzed by indirect immunofluorescent microscopy. One culture demonstrated defects of BP180 and LAD-1 expression (which was also verified by radioimmunoprecipitation assay), and one culture showed decreased laminin-5 expression but normal BP180 and LAD-1 expression. Thus, these studies demonstrate that: (i) LAD-1 and BP180 are normally expressed in all subtypes of JEB except GABEB, (ii) the majority of GABEB patients show absent or near absent expression of both LAD-1 and BP180 but normal expression of laminin-5, and (iii) a smaller subset of GABEB patients show normal LAD-1 and BP180 expression but express persistent but reduced levels of laminin-5.

Differential activation of protein kinase C isozymes by phorbol ester and collagen in human skin microvascular endothelial cells.

Human dermal microvascular endothelial cells participate in activities including inflammation, wound healing, and angiogenesis (neovascularization). Two stages of angiogenesis can be mimicked in vitro by two models of cultured foreskin human dermal microvascular endothelial cells: the differentiation of epithelioid endothelial cells to spindle-shaped mesenchymal-like cells induced by phorbol ester treatment; and the formation of vascular channels induced by exposing the luminal surface of endothelial cell monolayers to type I collagen gels. The mechanisms underlying these two processes, however, are largely unknown. Protein kinase C isozymes, which are activated by phorbol esters, are important mediators in the angiogenic process. In the current work, we identified the protein kinase C isozymes present in human dermal microvascular endothelial cells and determined which of the isozymes are activated in response to phorbol ester or to collagen treatments. Using western blot analysis, we found that microvascular endothelial cells contain at least six protein kinase C isozymes (alpha, beta, delta, epsilon, zeta, eta). Immunocytochemical studies demonstrated that the isozymes are located in distinct cellular compartments and that following treatment with phorbol 12-myristate 13-acetate or with a collagen gel overlay, most isozymes (protein kinase C alpha, beta1, betaII, delta, epsilon, eta) translocated to different parts of the cell. Moreover, for two of these isozymes (betaII and eta), the localization differs after phorbol 12-myristate 13-acetate treatment as compared with collagen treatment. These results demonstrate that agents that mimic two stages in the angiogenic process in vitro initiate diverse changes in the subcellular localization of specific protein kinase C isozymes and suggest a role for different isozymes in this process.

Gamma 2 chain of laminin-5 is recognized by monoclonal antibody GB3.

Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.

Benign lymphangioendothelioma.

We describe a 40-year-old white man with a red-brown, indurated plaque on the proximal aspect of his right thigh. The lesion had been present since birth, and the patient had a 20-year clinical history of recurrent cellulitis in the same area. The histopathologic features of the lesion included permeation of the dermis by flattened, endothelium-lined channels without cellular atypia, hemorrhage, or inflammation. The endothelial cells were stained intensely with monoclonal antibody anti-CD34 (clone MY10). In addition, antibodies to factor VIII antigen, HLA-DR, smooth muscle actin, ICAM-1, and the lectin Ulex europaeus labeled the luminal cells. The basement membrane of the channels stained with anti-type IV collagen and laminin. Desmin-positive cells were abundant adjacent to the channels. Factor XIIIa stained both mononuclear cells and occasional dendritic cells in the perivascular area. Ki-67 immunolabeling could not be demonstrated on fresh or frozen tissue. Electron microscopy revealed the presence of both tight junctions and a well-formed, continuous basement membrane but the absence of Weibel-Palade bodies.

Secreted inhibitors of metalloproteinases (IMPs) that are distinct from TIMP.

The tissue inhibitor of metalloproteinases (TIMP, M(r) 30,000) is secreted by many cell and tissue types and has been shown to inhibit most secreted mammalian metalloproteinases. In matrix and tissue invasion assays, the inactivation or removal of TIMP enhances invasiveness. However, many of the cells that secrete TIMP also secrete other metalloproteinase inhibitors. By analysis of medium conditioned by various endothelial, mesenchymal, and neural cells on SDS-.substrate-polyacrylamide-inhibitor gels (reverse zymograms), we have detected at least three other distinct inhibitors of metalloproteinases (IMPs). Some or all of these IMPs have been detected in secretions of mouse, rabbit, sheep, and human cells and are all smaller in apparent molecular size than TIMP (IMP-1, M(r) 26,000; IMP-2, M(r) 21,000; IMP-3, M(r) 18,000). These IMPs are not proteolytic degradation products of TIMP nor do they represent nonglycosylated TIMP. The IMPs do not cross-react in the native or denatured state with any of several anti-TIMP antibodies. The IMPs appear to be regulated independently of each other and of TIMP. In vitro, the complex consisting of one of the IMPs, or TIMP, and a metalloproteinase can be dissociated into functional inhibitor and metalloproteinase. Whether this characteristic is significant in vivo is not known. IMP-2 has been purified from several sources and shares sequence homology with TIMP, suggesting that the IMPs and TIMP may constitute a gene family. The most significant characteristic of IMP-2 is that it appears to preferentially inhibit, on a mole:mole basis, the M(r) 68,000 gelatinase rather than collagenase or stromelysin.(ABSTRACT TRUNCATED AT 250 WORDS)

Connective tissue proteinases and inhibitors in abdominal aortic aneurysms. Involvement of the vasa vasorum in the pathogenesis of aortic aneurysms.

Recent studies have shown that increases in proteolytic activity are associated with abdominal aortic aneurysms (AAAs). We have studied samples of the dilated aortic wall, taken during corrective surgery for AAAs, in terms of the number, type, and tissue location of connective tissue proteinases and their inhibitors. Five distinct caseinolytic serine proteinases and six gelatinolytic metalloproteinases were resolved by molecular weight by use of sodium dodecyl sulfate-substrate gel electrophoresis. Isoforms of the Mr 92,000 neutrophil gelatinase were identified by immunoprecipitation of biosynthetically labeled organ culture media. About 50% of the total radiolabeled protein secreted by AAA organ cultures was identified as the Mr 30,000 glycoprotein, tissue inhibitor of metalloproteinase (TIMP), by immunoprecipitation. Both TIMP and gelatinase were localized to the vasa vasorum by immunoperoxidase staining. However, interstitial collagenase could not be detected by any method. These results suggest the involvement of the vasa vasorum in the maintenance and possibly the genesis of AAAs.

Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity.

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.

Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors.

Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.