Human Renal Microvascular Endothelial Cells Research Articles

Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.

Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20–30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Proliferative potential of human kidney endothelial cells: bone marrow-derived cells may not be required for high proliferation

Proliferative potential of a single cell, defined as the number of progeny it gives rise to, has been used to define a hierarchy of endothelial progenitor cells in blood. Cells with high proliferative potential are presumed to have greater capacity for endothelium repair. Based on results with commercially available endothelial cells, it has been proposed that a proliferative hierarchy of endothelial cells also exists within blood vessels. It is unknown whether such vessel-derived highly proliferative endothelial cells originate from the bone marrow or whether the supply of precursors is limited to pre-existing cells that reside within vessels. In this study, we isolated normal human renal microvascular endothelial cells (RMEC) and larger cortical vessel endothelial cells (EC) by flow cytometry based on differential expression of human leucocyte antigen (HLA)-DR, and evaluated the proliferative potential of single cells. To determine if highly proliferative clones might derive from bone marrow recruits, HLA-DR expression on RMEC from transplanted kidneys was evaluated using antibodies that distinguish donor cells from recipient cells. We found the proliferative potential of kidney endothelial cells diverse and variable. Subcloning indicated that proliferative potential was determined by epigenetic events. In transplanted kidneys affected with a variety of different injuries, RMEC were donor derived. We conclude that endothelial cells of high proliferative potential exist within human renal blood vessels, even in individuals into their eighth decade of life, and that highly proliferative endothelial cells are unlikely to be bone marrow derived.

Proangiogenic Role of ephrinB1/EphB1 in Basic Fibroblast Growth Factor-Induced Corneal Angiogenesis

Corneal neovascularization is a vision-threatening condition caused by various ocular pathological conditions. The aim of this study was to evaluate the function of the ephrin ligands and Eph receptors in vitro and in vivo in corneal angiogenesis in a mouse model. The Eph tyrosine kinase receptors and their ligands, ephrins, are expressed on the cell surface. The functions of Eph and ephrins have been shown to regulate axonal guidance, segmentation, cell migration, and angiogenesis. Understanding the roles of Eph and ephrin in corneal angiogenesis may provide a therapeutic intervention for the treatment of angiogenesis-related disorders. Immunohistochemical studies demonstrated that ephrinB1 and EphB1 were expressed in basic fibroblast growth factor (bFGF)-induced vascularized corneas. EphB1 was specifically colocalized with vascular endothelial marker CD31 surrounded by type IV collagen. EphrinB1 was expressed in corneal-resident keratocytes and neutrophils. Recombinant ephrinB1-Fc, which induces EphB receptor activation, enhanced bFGF-induced tube formation in an in vitro aortic ring assay and promoted bFGF-induced corneal angiogenesis in vivo in a corneal pocket assay. Synergistically enhanced and sustained activation of extracellular signal-regulated kinase was noted in vascular endothelial cell lines after stimulation with ephrin B1 and bFGF combinations. These results suggest that ephrinB1 plays a synergistic role in corneal neovascularization.

Endothelial Microparticles (EMP) Bind and Complex with Unusually-Large Multimers of Von Willebrand Factor (ULvWf) and Act as Potent Inducers of Platelet Adhesion and Aggregation.

BACKGROUND. EMP have emerged as sensitive biomarkers of endothelial dysfunction. Little is known of their function. EMP interact with leukocytes and platelets and exert prothrombotic and proinflammatory effects. Recently we reported that vWf is expressed in EMP. The present study extends this observation to characterize EMP-bound vWf (EMP/vWf), specifically their multimer size and proaggregatory activity in ristocetin-induced platelet aggregation.METHODS. EMP, released from human renal microvascular endothelial cells 24hr following stimulation with TNF-α, were isolated by centrifugation (10,000g x 30min) and washed twice for vWf multimer analysis and proaggregatory activity assay. vWf multimer size was analyzed by 0.8% argarose gel electrophoresis and Western blotting. The proaggregatory activity of vWf was assessed flow ctyometrically by a two-step method: first, washed platelets were incubated with either EMP or soluble vWf such as plasma or Humate P (a commercial vWf multimers) in the presence of ristocetin for 10 min, then disappearance of free platelets as they formed small aggregates was measured. Second, the formed platelet aggregates were diluted 20-fold with buffer and degree of disaggregation was determined at intervals to assess the stability of PltAg.RESULTS. (i) Electrophoresis showed that vWf bands from normal plasma do not exceed 1,000 kDa and Humate P about 2,000 kDa while vWf bands on EMP extends much higher than 2,000 kDa. The protein banding pattern of EMP/vWf also differed from free soluble vWf (plasma) or humate P, the latter both showing clear ladder-like banding while EMP/vWf often appeared as a continuous smear of sizes in the high molecular weight regions, probably due to covalent bonds to transmembrane structures. (ii) Incubation of EMP (1 x 107 in 100 μL) with humate P (0.3 U) for 2hr (rotary shaker, 100 rpm) followed by washing the EMP revealed that significant additional vWf was adsorbed to the EMP: the vWf in the supernatant was decreased by 30% – 40%, and Western blotting showed additional high MW band on the treated EMP. This demonstrates that EMP express unoccupied sites capable of binding free vWf. (iii) Incubating EMP/vWf with washed platelets in the presence of ristocetin resulted in formation of platelet aggregates (PltAg). When PltAg were diluted 1:20, the PltAg began dissociating into free platelets in a time-dependent manner. The time for 50% dissociation of PltAg was 15–20min with soluble vWf (plasma or Humate P), but was prolonged to 45–60min with EMP/vWf. We attribute this mainly to the presence of ULvWf on EMP. (iv) Plasma from 4 acute TTP patients showed elevated EMP and enhanced formation of PltAg resistant to disaggregate. While 5 vWD plasma showed the opposite effect and their deficiency was corrected by added EMP.CONCLUSIONS. A new role of EMP is indicated in the present study. EMP-bound vWf is functionally active, inducing platelet aggregates of greater stability than free vWf in a ristocetin-based assay. EMP are capable of adsorbing additional vWf from plasma, indicating presence of extra binding sites. EMP enhance the stability of aggregates probably through ULvWf on the EMP, or to additional platelet-EMP-platelet adhesion molecules, or both. Through their high-affinity interaction with ULvWf, EMP likely play a role in vWf-dependent thrombotic disorders such as TTP and other platelet mediated thrombosis.

Tight Control of Platelet-derived Growth Factor B/c-sis Expression by Interplay between the 5′-Untranslated Region Sequence and the Major Upstream Promoter

The long and GC-rich 5'-untranslated region (5'-UTR) of the known 3.8-kb platelet-derived growth factor B (PDGF-B)/c-sis mRNA is highly conserved and inhibits its own translation. It has been thought that this 5'-UTR functions by regulating translation possibly using an internal ribosome entry site (IRES)-mediated mechanism. However, in the present study we found no evidence that the 5'-UTR sequence of PDGF-B mRNA contains any IRES activity. Instead, we found that the 5'-UTR sequence of PDGF-B functions as a promoter both constitutively and upon induction in a variety of cell lines. The 5'-UTR sequence contains two promoters (termed P1 and P2) when only the 5'-UTR sequence is analyzed. In the presence of the upstream TATA-box-containing promoter (P0), P1 and P0 promoters are integrated into one promoter, whereas the P2 promoter still functions. The full promoter with combined P0, P1, and P2 produced two transcripts, with the major one having the full-length 5'-UTR and the minor one the short 5'-UTR. The integrated P0/P1 promoter and P2 promoter are likely responsible for producing the endogenous 3.8- and 2.8-kb PDGF-B mRNAs that are detected in cultured human renal microvascular endothelial cells, a few tumor cells, and rat brain tissues. Furthermore, we detected the 2.8-kb PDGF-B mRNA in erythroleukemia K562 cells upon 12-O-tetradecanoylphorbol-13-acetate-induced differentiation. Considering that the 5'-UTR in the 3.8-kb mRNA contains no IRES activity and inhibits cap-dependent translation, we believe that the endogenous 2.8-kb mRNA produced from the 5'-UTR promoter is likely the major template responsible for protein production both constitutively and upon induction. We also found that the transcription from the 5'-UTR P2 promoter might be coordinated by the major upstream P0 promoter upon stimulation. Based on these observations, we propose that the TATA-containing P0 promoter and the 5'-UTR promoter work together to tightly control the expression of PDGF-B.

Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins.

Receptors of the Eph family and their ligands (ephrins) mediate developmental vascular assembly and direct axonal guidance. Migrating cell processes identify appropriate targets within migratory fields based on topographically displayed ephrin gradients. Here, EphB1 regulated cell attachment by discriminating the density at which ephrin-B1 was displayed on a reconstituted surface. EphB1-ephrin-B1 engagement did not promote cell attachment through mechanical tethering, but did activate integrin-mediated attachment. In endothelial cells, attachment to RGD peptides or fibrinogen was mediated through alphavbeta3 integrin. EphB1 transfection conferred ephrin-B1-responsive activation of alpha5beta1 integrin-mediated cell attachment in human embryonic kidney cells. Activation-competent but signaling-defective EphB1 point mutants failed to stimulate ephrin-B1 dependent attachment. These findings lead us to propose that EphB1 functions as a 'ligand density sensor' to signal integrin-mediated cell-matrix attachment.

Reduced extracellular pH increases endothelin-1 secretion by human renal microvascular endothelial cells.

Because dietary acid increases renal secretion of endothelin-1 (ET-1) which is synthesized by renal microvascular endothelium, we examined if reduced extracellular pH increases ET-1 secretion by cultured human renal microvascular endothelial cells (RMVECs). Confluent cells were exposed to serum-free media for 24 h, then incubated in either control, acid, or alkaline serum-free media for 12 h. Standard growth media pH was 7.2 after equilibration with 5% CO2 at 37 degrees C and was made the pH of control media. Acid and alkaline media pH were 7.0 and 7.4, respectively. Added Hepes and Tris maintained all assigned pHs. Media ET-1 measured by RIA after column extraction was higher for RMVECs exposed to acid compared with control media (170.0+/-17.1 vs. 64.6+/-9.6 pM, P < 0.004) but those exposed to alkaline media (56.6+/-25.1 pM, P = NS vs. control) were not. Human aortic endothelial cells exposed to control, acid, and alkaline media had similar ET-1 (166.6+/-18.1, 139.3+/-18.5, and 205.9+/-25.3 pM, P = NS). The data show acid-stimulated ET-1 secretion by RMVECs but not aortic endothelial cells, demonstrating a new environmental factor that influences ET-1 secretion by renal microvascular endothelium and thereby possibly modulates endothelin-dependent processes in vivo.

Identification of a subpopulation of human renal microvascular endothelial cells with capacity to form capillary-like cord and tube structures.

Endothelial specialization is a prominent feature within distinct capillary beds of organs such as mammalian kidney, yet immunological markers for functionally distinct subpopulations of cultured endothelial cells from tissue sources such as kidney have not been available. We developed a simple and reproducible isolation and culture procedure to recover human renal microvascular endothelial cells (HRMEC) from the cortex of unused donor kidneys. This procedure yields highly purified preparations of cells that display endothelial markers that include Factor VIII antigen, acetyl-LDL receptors, and determinants that bind Ulex europaeus lectin. HRMEC assemble into capillary-like cord and tube structures when plated on the surface of basement membrane-like matrix (BMM) in media containing phorbol myristate acetate. To further define subpopulations of HRMEC, we generated a panel of monoclonal antibodies and screened for those recognizing cell surface determinants. One monoclonal antibody recovery from this screen recognized a cell surface protein expressed on a subpopulation of HRMEC that we have designated PEC-1 (pioneer endothelial cell antigen-1). Cells expressing PEC-1 extended long, interconnecting filopodial processes in response to phorbol myristate acetate and assembled into capillary-like structures when plated on BMM. Anti-PEC-1 immunoprecipitated proteins of 25 and 27 kDa. Magnetic bead separation of PEC-1 (+) cells selected cells that assemble into capillary-like cord and tube structures. The remaining PEC-1 (-) HRMEC population formed matrix adherent patches. In the kidney, the PEC-1 determinant is expressed on a small subpopulation of microvascular glomerular cells and is prominently expressed on the apical membrane of proximal tubule cells. The PEC-1 determinant discriminates among subpopulations of HRMEC, identifying a subpopulation that contributes to assembly of capillary-like structures.

Ligand Activation of ELK Receptor Tyrosine Kinase Promotes Its Association with Grb10 and Grb2 in Vascular Endothelial Cells

ELK is a member of the Eph-related tyrosine kinase family that includes receptors signaling axonal guidance, neuronal bundling, and angiogenesis. We recently identified ELK expression in human renal microvascular endothelial cells and sought to identify intracellular proteins through which it signals responses. The cytoplasmic domain of ELK was used as "bait" in a yeast two-hybrid screen to identify interactive proteins expressed from a randomly primed embryonic murine library (E9.5-10.5). Among interactive products of 76 cDNAs characterized, 10 nonidentical, overlapping clones encoded the SH2 domain of the recently reported Grb10 adapter protein, and an additional 3 encoded Grb2. A self-phosphorylated recombinant, baculovirus-expressed GST-ELKcy fusion protein bound Grb10 and Grb2 from human renal microvascular endothelial cell extracts, while the unphosphorylated fusion form did not. Site-directed mutation identified Tyr-929 as a putative phosphorylation site required for Grb10, but not Grb2, interaction in yeast and recombinant protein assays. The ELK ligand, LERK-2/Fc, stimulated tyrosine phosphorylation of ELK, and recruitment of Grb10 and Grb2 to endothelial ELK receptors recovered by wheat germ agglutinin lectin and immunoprecipitation. These findings define ligand-activated interaction between ELK and the SH2 domains of Grb2 and the newly identified Grb10 protein that shares homology with a Caenorhabditis elegans gene product implicated in neural cell migration.

Human Renal Microvascular Endothelial Cells as a Potential Target in the Development of the Hemolytic Uremic Syndrome as Related to Fibrinolysis Factor Expression, in Vitro

Renal glomerular microvascular endothelial cell damage is characteristic of Shiga toxin-associated hemolytic uremic syndrome (HUS). An impaired renal fibrinolysis may be responsible for renal microvascular fibrin accumulation during the course of HUS disease. This study examined the effect of Shiga toxin, bacterial lipopolysaccharide (LPS, endotoxin), and tumor necrosis factor (TNF) on the expression of fibrinolysis factors by human renal glomerular microvascular endothelial cells (HRMEC) in vitro. The results were compared to a previously better-characterized endothelial cell type, human umbilical vein endothelial cells (HUVEC). In HUVEC, the ratio of fibrinolysis antigens was antifibrinolytic, consisting of 55-fold more plasminogen activator inhibitor type 1 (PAI-1) than tissue-type plasminogen activator (tPA). Treatment of HUVEC with LPS or TNF accentuated this ratio by decreasing tPA and increasing PAI-1 expression. In contrast, HRMEC produced urokinase-type plasminogen activator (uPA) in a 24-fold excess to PAI-1 and were thereby profibrinolytic with regard to fibrinolysis antigen expression. LPS and TNF further decreased PAI-1 antigen expression by HRMEC. These results argue against a role for LPS or TNF in decreasing renal fibrinolysis at the level of fibrinolysis factor expression by renal endothelial cells. Nevertheless, HUVEC and HRMEC were responsive to the same LPS analogs in the same order of potency. Shiga toxin decreased fibrinolysis factor expression to a greater extent in HRMEC than in HUVEC. Since HRMEC fibrinolysis antigen expression was profibrinolytic, the Shiga toxin-mediated decrease in renal endothelial uPA synthesis may predispose renal microvasculature to thrombosis and may have implications for the development of HUS.

A western blot assay detects autoantibodies to cryptic endothelial antigens in thrombotic microangiopathies

Autoantibodies detected by immunofluorescence, ELISA, and complement-fixation techniques have provided discriminatory markers for many human diseases. However, these commonly applied assays may fail to detect antibodies against antigenic sites which are either inaccessible or not displayed in recognizable cellular structures. Moreover, molecular identities of recognized antigen(s) are not determined with such methods. We have used Western blot analysis of cellular proteins derived from human renal microvascular endothelial cells (HRMEC) to identify autoantibodies in patients with pathological endothelial injury. Exploring the possibility that endothelial injury may expose cryptic endothelial antigens to immune recognition, we detected antibodies binding a number of distinct HRMEC proteins. Among these, antibodies recognizing specific HRMEC proteins of 43 kDa were commonly detected in plasmas from patients with thrombotic thrombocytopenic purpura (TTP) (13 of 14) and hemolytic uremic syndrome (HUS) (4 of 5) but were absent in 9 of 10 healthy subjects and 11 patients with a range of diseases not associated with endothelial injury or insult. Antibodies binding 43-kDa HRMEC antigens were detected in individual patients with systemic lupus erythematosus, anti-glomerular basement membrane nephropathy, and heparin-associated thrombocytopenia, as well as in one of three patients with immune thrombocytopenic purpura. Similar antibodies were detected in one hypercholesterolemic subject. Antibodies from four TTP patients were affinity purified and shown by two-dimensional analysis to recognize 43-kDa proteins having identical pl's (5.9, 6.0, and 6.1). Subcellular fractionation localized these antigens to cytosolic and nuclear compartments, sites presumably protected from immune recognition in the absence of endothelial injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct pathways mediate transcriptional regulation of platelet-derived growth factor B/c-sis expression.

The biochemical mechanisms responsible for regulating cellular platelet-derived growth factor expression are incompletely understood. Our previous studies have shown that platelet-derived growth factor B/c-sis mRNA levels are induced in human renal microvascular endothelial cells by either thrombin or transforming growth factor (TGF-beta), while exposure to agents which elevate cAMP levels blocks the induction responses. The current studies use combined transcription run-off and message decay rate experiments to show greater than 3-fold increases in rate of transcription after stimulation with either thrombin or TGF-beta. c-sis message has a 70-90-min half-life under basal conditions that is effectively unaltered by thrombin or TGF-beta. Forskolin does not decrease the stability of c-sis mRNA, although it attenuates transcription increases seen with inducing agents. TGF-beta induction of c-sis transcription is mediated independent of the protein kinase C (Ca2+- and phospholipid-dependent enzyme)-mediated responses to phorbol ester, as it remains intact following down-regulation of protein kinase C response; TGF-beta and phorbol elicit additive induction. Inhibitory effects of cAMP upon transcription act distal to early thrombin-receptor-coupled increases in phosphatidylinositol turnover and are capable of turning off TGF-beta-activated transcription after activation has been established. Both inducing and suppressing agents alter endothelial platelet-derived growth factor B/c-sis mRNA expression dominantly through effects upon rates of transcription, cAMP suppression of transcription is dominant, and TGF-beta and phorbol esters mediate induction of transcription through distinct pathways.