Rat Endothelium Research Articles

NDRG1 promotes endothelial dysfunction and hypoxia-induced pulmonary hypertension by targeting TAF15.

Pulmonary hypertension (PH) represents a threatening pathophysiologic state that can be induced by chronic hypoxia and is characterized by extensive vascular remodeling. However, the mechanism underlying hypoxia-induced vascular remodeling is not fully elucidated. By using quantitative polymerase chain reactions, western blotting, and immunohistochemistry, we demonstrate that the expression of N-myc downstream regulated gene-1 (NDRG1) is markedly increased in hypoxia-stimulated endothelial cells in a time-dependent manner as well as in human and rat endothelium lesions. To determine the role of NDRG1 in endothelial dysfunction, we performed loss-of-function studies using NDRG1 short hairpin RNAs and NDRG1 over-expression plasmids. In vitro, silencing NDRG1 attenuated proliferation, migration, and tube formation of human pulmonary artery endothelial cells (HPAECs) under hypoxia, while NDRG1 over-expression promoted these behaviors of HPAECs. Mechanistically, NDRG1 can directly interact with TATA-box binding protein associated factor 15 (TAF15) and promote its nuclear localization. Knockdown of TAF15 abrogated the effect of NDRG1 on the proliferation, migration and tube formation capacity of HPAECs. Bioinformatics studies found that TAF15 was involved in regulating PI3K-Akt, p53, and hypoxia-inducible factor 1 (HIF-1) signaling pathways, which have been proved to be PH-related pathways. In addition, vascular remodeling and right ventricular hypertrophy induced by hypoxia were markedly alleviated in NDRG1 knock-down rats compared with their wild-type littermates. Taken together, our results indicate that hypoxia-induced upregulation of NDRG1 contributes to endothelial dysfunction through targeting TAF15, which ultimately contributes to the development of hypoxia-induced PH.

Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury.

Maintaining the integrity of the blood-spinal cord barrier is critical for the recovery of spinal cord injury. Ferroptosis contributes to the pathogenesis of spinal cord injury. We hypothesized that ferroptosis is involved in disruption of the blood-spinal cord barrier. In this study, we administered the ferroptosis inhibitor liproxstatin-1 intraperitoneally after contusive spinal cord injury in rats. Liproxstatin-1 improved locomotor recovery and somatosensory evoked potential electrophysiological performance after spinal cord injury. Liproxstatin-1 maintained blood-spinal cord barrier integrity by upregulation of the expression of tight junction protein. Liproxstatin-1 inhibited ferroptosis of endothelial cell after spinal cord injury, as shown by the immunofluorescence of an endothelial cell marker (rat endothelium cell antigen-1, RECA-1) and ferroptosis markers Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Liproxstatin-1 reduced brain endothelial cell ferroptosis in vitro by upregulating glutathione peroxidase 4 and downregulating Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase. Furthermore, inflammatory cell recruitment and astrogliosis were mitigated after liproxstatin-1 treatment. In summary, liproxstatin-1 improved spinal cord injury recovery by inhibiting ferroptosis in endothelial cells and maintaining blood-spinal cord barrier integrity.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01−100 μM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 μM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 μM), the DP1 receptor antagonist BW A868C (1 μM) also inhibited relaxation reaching significance above 10 μM. In contrast, the EP3 receptor antagonist L798106 (1 μM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 μM) blocked transforming growth factor β1 (TGF-β1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 μM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-β1 in the presence of TRE.

Augmentation of Hyperglycemia‐Induced Damage in a Diabetic Rat Endothelium is Potentially Regulated by the Nfκβ Signaling Pathway

Endothelial dysfunction is a key factor for the development of organ failure in type 2 diabetes mellitus (T2DM). While hyperglycemia is a well‐established factor in the development of microvascular complications in T2DM, specific mechanisms of endothelial damage remains equivocal. In a previous study, we observed that hyperglycemic states resulted in distinct alterations in proteins involved in metabolic and angiogenic processes in endothelial cells isolated from diabetic when compared to non‐diabetic rats. In the present study, we identified and validated diverse NFκβ‐regulated cellular functions that are involved in augmenting hyperglycemia‐induced damage in the diabetic endothelium. Bioinformatics and functional studies were carried out in the primary cardiac microvascular endothelial cells (RCMVECs) derived from the spontaneously T2DM Goto‐Kakizaki (GK) rat model in comparison to the control Wistar Kyoto (WKY) rat model under normal and hyperglycemic conditions. GK and WKY RCMVECs were cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks, followed by tandem mass spectrometry (MS/MS), qPCR, western blotting, microplate‐based cell proliferation, metabolic and REDOX assays. Evidence of hyperglycemia‐induced cytotoxicity in GK RCMVECs was determined by employing various microplate‐based assays, wherein a 20% reduction in cell proliferation (p<0.05) and a 33% increase in PMA‐induced ROS production (p<0.05) was observed. Enrichment and pathway analyses of the proteomic tandem MS/MS and qPCR datasets, revealed induction of inflammatory, metabolic and apoptotic pathways, which are tightly regulated by NFκβ, in GK RCMVECs (HG vs NG). Platelet‐activating factor receptor (LogFC = 3.5, p <0.01) and beta‐catenin (LogFC= 1.3, p <0.01), both key inductors of NFκβ signaling, were significantly upregulated in GK RCMVECs (HG vs NG). Western blotting analysis revealed significant upregulation of NFκβ in GK RCMVECs (HG vs NG) (FC=1.6, p<0.05) and in GK NG vs WKY NG RCMVECs (FC=2.5, p<0.05), suggesting a potential increase in NFκβ activity in the diabetic endothelium. Additionally, the activity of hexokinase‐2, which is reported to be a transcriptional target of NFκβ, was observed to be reduced in GK NG vs WKY NG RCMVECs (20% reduction, p<0.05). Furthermore, qPCR analysis identified an upregulation of several inflammatory transcripts that are that are regulated by NFκβ such as CXCR4 (FC=7.5, p<0.01) and NLRP3 (FC=5.8, p<0.01) expression in GK RCMVECs (HG vs NG). By the integration of high‐throughput tandem MS/MS analysis, bioinformatics analyses and in vitro functional assays, we identified a potential induction of NFκβ signaling in GK (HG vs NG) RCMVECs, resulting in metabolic reprogramming and a heightened inflammatory and apoptotic response.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.

Protective effect of thymoquinone on smoking-induced vascular damage: An experimental study in rats

Aim: To investigate the protective effect of thymoquinone on smoking-induced vascular damage in rats. Method: A total of 28 rats were allocated in this experimental study. Rats were equally divided in four groups; as control group (n=7) and study groups including only smoking group (n=7), smoking plus thymoquinone group (n=7) and smoking plus dexamethasone group (n=7). The animals in study groups were subjected to regular cigarette smoke exposure during 2 months, hereby smoking-induced vascular injury model was established in the animals. The thoracic aorta of the animals were surgically resected and then evaluated histopathologically. The prepared aortic tissue samples were analyzed under light microscope in terms of thickness of aortic wall, endothelial continuity and injury as well as degenerative alterations on the endothelium. Results: Mean aortic wall thicknesses were 77.8 and 114.5 μ in only smoking group and smoking plus thymoquinone group respectively, and this difference was statistically significant. In only smoking group partial endothelial damage and complete endothelial damage were observed in 2 and 4 animals respectively whereas in smoking plus thymoquinone group no endothelial damage and partial endothelial damage were observed in 2 and 4 animals respectively. When these groups were compared in terms of endothelial damage, the difference was detected to be statistically significant. Conclusion: Our study demonstrates that thymoquinone has a protective effect on rat endothelium and alleviates the smoking-induced vascular damage in rats.

Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells.

Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5 pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function.

Gender specific generation of nitroxyl (HNO) from rat endothelium

Nitric oxide (NO) has long been accepted as the majority biological mediator underlying the critically important vasodilator function of the vascular endothelium yet there is growing evidence that the protonated one-electron reduction species of NO, nitroxyl (HNO), may also have biological activity. In this study we examined if there was a gender variation in the production of HNO from the endothelium of isolated segments of rat aorta. By use of recognized pharmacological inhibitors, we found that when the endothelium was stimulated by acetylcholine vascular relaxation was mediated entirely via NO in tissue from both males and females. The vasorelaxation induced by basal (non-stimulated) endothelium from females was also mediated entirely by NO. However, in contrast, the influence of basally active endothelium in males was mediated by both NO and HNO. The generation of HNO in males was dependent on endothelial nitric oxide synthase and relaxation was mediated via activation of soluble guanylate cyclase. We believe that this is the first evidence of a gender variation in the production of HNO and this may have important implications for our understanding of disparity in the development of cardiovascular disease between the sexes.

Loss of Bone Morphogenetic Protein Receptor 2 Is Associated with Abnormal DNA Repair in Pulmonary Arterial Hypertension

Occlusive vasculopathy with intimal hyperplasia and plexogenic arteriopathy are severe histopathological changes characteristic of pulmonary arterial hypertension (PAH). Although a phenotypic switch in pulmonary endothelial cells (ECs) has been suggested to play a critical role in the formation of occlusive lesions, the pathobiology of this process is poorly understood. The goal of this study was to identify novel molecular mechanisms associated with EC dysfunction and PAH-associated bone morphogenetic protein receptor 2 (BMPR2) deficiency during PAH pathogenesis. A bioinfomatics approach, patient samples, and in vitro experiments were used. By combining a metaanalysis of human idiopathic PAH (iPAH)-associated gene-expression microarrays and a unique gene expression-profiling technique in rat endothelium, our bioinformatics approach revealed a PAH-associated dysregulation of genes involving chromatin organization, DNA metabolism, and repair. Our hypothesis that altered DNA repair and loss of genomic stability play a role in PAH was supported by in vitro assays where pulmonary ECs from patients with iPAH and BMPR2-deficient ECs were highly susceptible to DNA damage. Furthermore, we showed that BMPR2 expression is tightly linked to DNA damage control because excessive DNA damage leads to rapid down-regulation of BMPR2 expression. Moreover, we identified breast cancer 1 (BRCA1) as a novel target for BMPR2 signaling and a novel modulator of pulmonary EC homeostasis. We show here that BMPR2 signaling plays a critical role in the regulation of genomic integrity in pulmonary ECs via genes such as BRCA1. We propose that iPAH-associated EC dysfunction and genomic instability are mediated through BMPR2 deficiency-associated loss of DNA damage control.

Hypercholesterolaemia, signs of islet microangiopathy and altered angiogenesis precede onset of type 2 diabetes in the Goto–Kakizaki (GK) rat

The adult non-obese Goto-Kakizaki (GK) rat model of type 2 diabetes, particularly females, carries in addition to hyperglycaemia a genetic predisposition towards dyslipidaemia, including hypercholesterolaemia. As cholesterol-induced atherosclerosis may be programmed in utero, we looked for signs of perinatal lipid alterations and islet microangiopathy. We hypothesise that such alterations contribute towards defective pancreas/islet vascularisation that might, in turn, lead to decreased beta cell mass. Accordingly, we also evaluated islet inflammation and endothelial activation in both prediabetic and diabetic animals. Blood, liver and pancreas were collected from embryonic day (E)21 fetuses, 7-day-old prediabetic neonates and 2.5-month-old diabetic GK rats and Wistar controls for analysis/quantification of: (1) systemic variables, particularly lipids; (2) cholesterol-linked hepatic enzyme mRNA expression and/or activity; (3) pancreas (fetuses) or collagenase-isolated islet (neonates/adults) gene expression using Oligo GEArray microarrays targeted at rat endothelium, cardiovascular disease biomarkers and angiogenesis, and/or RT-PCR; and (4) pancreas endothelial immunochemistry: nestin (fetuses) or von Willebrand factor (neonates). Systemic and hepatic cholesterol anomalies already exist in GK fetuses and neonates. Hyperglycaemic GK fetuses exhibit a similar percentage decrease in total pancreas and islet vascularisation and beta cell mass. Normoglycaemic GK neonates show systemic inflammation, signs of islet pre-microangiopathy, disturbed angiogenesis, collapsed vascularisation and altered pancreas development. Concomitantly, GK neonates exhibit elevated defence mechanisms. These data suggest an autoinflammatory disease, triggered by in utero programming of cholesterol-induced islet microangiopathy interacting with chronic hyperglycaemia in GK rats. During the perinatal period, GK rats show also a marked deficient islet vascularisation in conjunction with decreased beta cell mass.

PP111 Examination of the expression profiles of PGIS and TXS in NSCLC: Regulation of tumor cell growth and invasive potential

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF1α and TXB2 aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF 1α and TXB 2 aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.

Evidence for a functional role of endothelial TRPV4 in shear stress‐induced vasodilatation

Ca2+-influx through TRP channels is believed to play a role in the synthesis of endothelial vasodilators. Here, we investigated function and expression of the TRPV4 channel in endothelial cells (EC) by using the in situ patch-clamp technique, single-cell-RT-PCR, Ca2+-imaging, and pressure-myograph experiments. In EC in situ, TRPV4-like currents were activated by the selective TRPV4-opener 4α-phorbol-12,13-didecanoate (4αPDD), arachidonic acid (AA), moderate warmth, and hypotonic stress. Single-cell-RT-PCR revealed expression of TRPV4, but none of the other closely related TRPV1-3. Activation of this endothelial TRPV4 by 4αPDD increased intracellular [Ca2+]i to 250 nM. In pressure-myograph experiments in carotid artery (CA) and small A. gracilis, intraluminal application of 4αPDD caused a robust vasodilatation by ≈80% (at 1 uM; KD 0.3 uM) which was strictly endothelium-dependent and was suppressed by the TRPV4-inhibitor ruthenium red (RuR). Wall shear stress (WSS)-induced vasodilatation was similarly blocked by RuR and was also prevented by inhibition of phospholipase A2 by AACOCF3. In the presence of inhibitors of NO- and prostacyclin-synthesis, 4αPDD and WSS did not produce considerable vasodilatation of CA whereas 4αPDD but not WSS caused vasodilatation of small A. gracilis. In conclusion, Ca2+-entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation in rat endothelium. Since blockage of TRPV4 suppressed WSS-induced vasodilatation, TRPV4 seems to play a role in endothelial mechanosensing of WSS.

Inhibiting Binding of Sickle Red Cell ICAM-4 to Endothelial Cell αVβ3 Integrin Decreases Red Cell Adhesion and Vaso-Occlusion.

Convincing evidence is accumulating that in sickle cell disease adhesion molecules on sickle red cells (RBCs) contribute to increased interactions with vascular endothelial cells leading to vaso-occlusion. Erythrocyte ICAM-4, a member of the immunoglobulin superfamily, binds multiple integrins including αVβ3. Earlier investigations showed that antibodies to αVβ3 inhibit human sickle RBC adherence to rat endothelium and improve microvascular dynamics, suggesting that αVβ3 may be an endothelial cell integrin functioning in vaso-occlusion. More recent in vitro studies revealed that epinephrine can act via a PKA-dependent pathway to activate ICAM-4 mediated sickle RBC adhesion to endothelial cell αVβ3. We are exploring the contribution of ICAM-4/αV interactions to vaso-occlusion utilizing a well established ex vivo mesocecum vasculature model that enables intravital microscopic observation and quantitation of sickle RBC adhesion to endothelial cells in the microcirculation, as well as hemodynamic measurements. A molecular model of ICAM-4 presents the extracellular region as two Ig-like domains comprised of A,B,C,D,E,F, and G strands. Earlier, we identified a patch or footprint on ICAM-4 comprised of three A strand residues and five G strand residues, that mediates adhesion to αV. In the current studies we quantitated sickle RBC adhesion to endothelial cells in the presence of two synthetic peptides that block ICAM-4/αV adhesion, peptides FWV and ATSR which correspond to sequences of the A and G strands of ICAM-4 domain 1, respectively. Sickle RBCs were obtained from stable HbSS patients who had not been transfused for at least 4 months and who were not receiving hydroxyurea. Isolated rat mesocecum was perfused with platelet-activating factor (PAF), that enhances sickle RBC adhesion. Then peptide FWV or ATSR (250 μMol/L) was infused, and following a 30 minute incubation, a bolus of washed, oxygenated sickle RBCs (Hct 30%) was perfused at a pressure of 60 mm Hg. Peptides were each tested with cells obtained from the same patients. Quantitation of adherent sickle RBCs per 100 μm2 in the affected venules was determined by direct microscopic observations and video analysis. In preparations treated with PAF alone, adhesion of sickle RBCs showed a strong inverse correlation with venular diameter. Adhesion was maximal in small-diameter venules, frequently resulting in vessel blockage. In preparations treated with peptide FWV, sickle RBC adhesion was distinctly decreased in venules of all diameters. ATSR resulted in even more pronounced inhibition of RBC adhesion, particularly in small-diameter venules. Comparison of regression lines revealed significantly lower Y-intercepts for both FWV and ATSR as compared with PAF-treated controls (p<0.00001). With both FWV and ATSR no blockage was observed in postcapillary venules. In marked contrast, a strand F control peptide, AWSS, had minimal effect. Our data greatly strengthen the thesis that N-terminal domain 1 of ICAM-4 on sickle RBCs binds endothelial cells via αVβ3 and that this interaction significantly contributes to vaso-occlusion in sickle cell disease. These findings suggest that peptides or small molecule mimetics of ICAM-4 may have potential as therapeutic agents.

The protective effect of protein kinase C and adenosine triphosphate-sensitive potassium channel agonists against inflammation in rat endothelium and vascular smooth muscle in vitro and in vivo.

Volatile anesthetic pretreatment protects the vasculature from inflammation-induced injury via mechanisms involving the activation of adenosine triphosphate-sensitive potassium (K(ATP)) channels and/or protein kinase C (PKC). Therefore, we hypothesized that K(ATP) and PKC agonists may mimic the protective effects of volatile anesthetics in vitro and in vivo. In vitro, rat vascular smooth muscle cells (VSM) and aortic endothelial cells (AEC) were used to evaluate whether pretreatment with a K(ATP) agonist, cromakalim (CRK), or a PKC agonist, phorbol 12-myristate 13-acetate (PMA), decreases lipopolysaccharide (LPS)-induced cell injury. Cell survival was determined by trypan blue staining after 6 h. In vivo, rats received systemic LPS or saline with or without pretreatment with PMA or CRK. Mean arterial blood pressure, the response to endothelium-dependent (acetylcholine; ACH) and -independent (sodium nitroprusside) vasodilators, and arterial blood gases were determined after 6 h. Cell survival in VSM and AEC control cultures was more than 90%, which was not altered in the presence of PMA or CRK, whereas LPS significantly decreased cell survival. PMA (0.1-10 microM) significantly attenuated the LPS-induced decrease in cell survival by 28%-37% in VSM and 39%-53% in AEC, and CRK (1 mM) increased cell survival by 24% in VSM and 22% in AEC. In vivo, PMA and CRK pretreatment had no significant effect on measured variables in control rats. LPS decreased mean arterial blood pressure and vasodilation to ACH and sodium nitroprusside and caused hypoglycemia. PMA, but not CRK, increased ACH-dependent vasodilation (46%) at 6 h, but neither agonist altered the other detrimental effects of LPS. In conclusion, PKC and K(ATP) agonists appear to protect AEC and VSM cells against inflammation in vitro, but the systemic administration of PKC and K(ATP) agonists appeared to exert minimal or no protection in our in vivo model.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.

IONIC AND NON-IONIC CONTRAST AGENT-MEDIATED ENDOTHELIAL INJURY. Quantitative analysis of cell proliferation during endothelial repair

Purpose: To quantitatively evaluate endothelial injury in vivo and to assess the time course of cellular repair after endothelial cell exposure to ionic and non-ionic contrast media. Material and Methods: Local changes at the cellular level following intraaortic injection of 1 ml of the ionic contrast agent ioxithalamate or 1 ml of the non-ionic contrast agent iomeprol, each with an iodine content of 300 mg/ml, were investigated using rat endothelium as an in vivo model. A sorbitol solution iso-osmolar to iomeprol served as control. Quantitative analysis of endothelial changes by autoradiography of 3H-thymidine-labeled endothelial cells was assessed after 3, 5 and 10 days, determining the 3H-thymidine index and the DNA synthesis rate. Results: Ioxithalamate showed a clear harmful effect on the endothelium, with an elevated 3H-thymidine index of 7.68% on day 3 and 6.89% on day 5, versus 2.97% on day 3 and 2.55% on day 5 for iomeprol and 2.29% on day 3 and 1.91% on day 5 for the control. Conclusion: Ionic contrast agents lead to reversible transient focal endothelial cell injury. No such side effect was detectable for non-ionic contrast agents.

Ionic and non-ionic contrast agent-mediated endothelial injury: Quantitative analysis of cell proliferation during endothelial repair

To quantitatively evaluate endothelial injury in vivo and to assess the time course of cellular repair after endothelial cell exposure to ionic and non-ionic contrast media. Local changes at the cellular level following intraaortic injection of 1 ml of the ionic contrast agent ioxithalamate or 1 ml of the non-ionic contrast agent iomeprol, each with an iodine content of 300 mg/ml, were investigated using rat endothelium as an in vivo model. A sorbitol solution iso-osmolar to iomeprol served as control. Quantitative analysis of endothelial changes by autoradiography of 3H-thymidine-labeled endothelial cells was assessed after 3, 5 and 10 days, determining the 3H-thymidine index and the DNA synthesis rate. Ioxithalamate showed a clear harmful effect on the endothelium, with an elevated 3H-thymidine index of 7.68% on day 3 and 6.89% on day 5, versus 2.97% on day 3 and 2.55% on day 5 for iomeprol and 2.29% on day 3 and 1.91% on day 5 for the control. Ionic contrast agents lead to reversible transient focal endothelial cell injury. No such side effect was detectable for non-ionic contrast agents.

Parathyroid-induced angiogenesis is VEGF-dependent

Background. Autotransplantation of parathyroid tissue after parathyroidectomy is successful at salvaging parathyroid function. The relatively high success of parathyroid transplantation is thought to be due, in part, to the ability of parathyroid tissue to induce angiogenesis and thus recruit a new vasculature. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor produced by a number of tumors and hypoxic tissues. Using a 3-dimensional intact microvessel angiogenesis system, we evaluated the role of VEGF in the stimulation of angiogenesis by human parathyroid cells. Methods. Freshly isolated rat microvessels embedded in a 3-dimensional collagen I matrix were treated with healthy 1-mm3 fragments of human parathyroid tissue or isolated parathyroid cells. Other gels were supplemented with VEGF165 or FLT-1 soluble receptor fusion protein to bind VEGF. After 11 days in culture, the gels were stained with Gs-1 lectin, a marker for rat endothelium, and linear growth of the microvessels was determined by using image analysis. Parathyroid production of VEGF was determined with reverse transcriptase-polymerase chain reaction. Results. A significant increase in microvessel growth was seen in parathyroid coculture (8.4 ± 1.0 mm) versus VEGF165 supplemented gels (6.2 ± 0.3 mm, P <.01). VEGF165 significantly augmented parathyroid-stimulated angiogenesis (13.7 ± 2.4 mm, P <.05 vs parathyroid alone). Using quantitative reverse transcriptase-polymerase chain reaction, we identified VEGF messenger RNA (mRNA) induction within 1 hour of parathyroid explant, with a 12-fold increase by 24 hours. Treatment of parathyroid cocultures with 0.2 μg/mL FLT-1 soluble receptor protein completely eliminated the parathyroid induction of angiogenesis. Conclusions. Parathyroid tissue expresses low levels of VEGF mRNA, which is significantly upregulated on explantation. Furthermore, the increased VEGF expression is essential to drive parathyroid-induced angiogenesis in our model. However, our data suggests that other parathyroid-produced factors are involved in mediating parathyroid-induced angiogenesis. (Surgery 2000;128:458–64)

Magnesium removal impairs the regulatory role of rat endothelium.

Vascular endothelium has been shown to play an important role in the regulation of vascular tone and, hence, impairment of the endothelium may induce hypertension. Although magnesium (Mg) deficiency could induce hypertension, the role of Mg on the endothelium is unclear. We examined the effects of Mg removal on endothelium-dependent and -independent responses using ring preparations of femoral arteries obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Norepinephrine (10(-9)-10(-4) M) evoked concentration-dependent contractions in arteries with endothelium. The maximal response was greater in SHR than in WKY. Removal of external Mg augmented the contraction in WKY but not in SHR. As a result, the contraction obtained in arteries with endothelium was identical in the two groups. Removal of the endothelium enhanced the contraction in both strains, with a greater response occurring in WKY than in SHR in Krebs, but not in Mg-free, solution. As a result, in arteries without endothelium, the contractions were identical in WKY and SHR both in Krebs and Mg-free solutions. Acetylcholine (10(-9)-10(-4) M) evoked concentration-dependent relaxation in arteries with, but not in those without, endothelium obtained from WKY and SHR. The relaxation did not differ between the two strains, nor was it altered by Mg removal. Thus, Mg removal impairs inhibitory function of the endothelium against contraction induced by norepinephrine, without affecting endothelium-dependent relaxation in response to acetylcholine, in the rat femoral artery. The effect of Mg removal is not apparent in SHR. The fact that after removal of external Mg the contraction in response to norepinephrine in arteries with endothelium is identical in WKY and SHR suggests that a normotensive artery with Mg deficiency may mimic a hypertensive artery through endothelial impairment.

Tyrosine Phosphorylation of Caveolin-1 in the Endothelium

Caveolin-1, a scaffolding protein of caveolae, is known to be tyrosine-phosphorylated by Src kinases. Recently we generated a specific antibody to caveolin-1 phosphorylated at tyrosine-14 (PY14) (R. Nomura and T. Fujimoto, 1999, Mol. Biol. Cell 10, 975–986). In the present study, by applying PY14 to sections of normal rat tissues, we found that tyrosine phosphorylation of caveolin-1 occurred in limited locations, including the endothelium of the continuous capillaries and small venules. Cultured endothelial cells were not labeled by PY14 under a standard culture condition, but became positively labeled when exposed to oxidative stresses and/or tyrosine phosphatase inhibitors. The reaction was prohibited by pretreating the cells with herbimycin A or genistein. Vasoactive reagents or physical stimuli did not cause the phosphorylation. Concomitant with the tyrosine phosphorylation, the number of invaginated caveolae decreased drastically, and vesicles labeled intensely for caveolin-1 appeared in the cytoplasm; the average diameter of the vesicles was larger than that of caveolae. The result implies that tyrosine phosphorylation of caveolin-1 occurs at tyrosine-14 in the normal rat endothelium in vivo and may induce caveolar vesiculation and/or fusion.