Primary Cardiac Endothelial Cells Research Articles

Endothelial FOSL1 drives angiotensin II-induced myocardial injury via AT1R-upregulated MYH9.

Vascular remodeling represents a pathological basis for myocardial pathologies, including myocardial hypertrophy and myocardial infarction, which can ultimately lead to heart failure. The molecular mechanism of angiotensin II (Ang II)-induced vascular remodeling following myocardial infarction reperfusion is complex and not yet fully understood. In this study, we examined the effect of Ang II infusion on cardiac vascular remodeling in mice. Single-cell sequencing showed Ang IIinduced cytoskeletal pathway enrichment and that FOS like-1 (FOSL1) affected mouse cardiac endothelial dysfunction by pseudotime analysis. Myosin heavy chain 9 (MYH9) was predominantly expressed in primary cardiac endothelial cells. The Ang II type I receptor blocker telmisartan and the protein kinase C inhibitor staurosporine suppressed Ang II-induced upregulation of MYH9 and FOSL1 phosphorylation in human umbilical vein endothelial cells. Silencing MYH9 abolished Ang II-mediated inhibition of angiogenesis in human umbilical vein endothelial cells, and attenuated AngII-induced vascular hyperpermeability. We found that FOSL1 directly bound to the MYH9 promoter and thus activated transcription of MYH9 by the dual luciferase reporter and chromatin immunoprecipitation assays, leading to vascular dysfunction. In vivo, 6 weeks after injecting adeno-associated virus-ENT carrying the TEK tyrosine kinase (tie) promoter-driven short hairpin RNA for silencing FOSL1 (AAV-tie-shFOSL1), cardiac function represented by the ejection fraction and fractional shortening was improved, myocardial fibrosis was decreased, protein levels of phosphorylated FOSL1, MYH9, and collagen type I alpha were reduced, and cardiac vascular density was recovered in mice with endothelial Fosl1-specific knockdown in Ang II-infused mice. In ischemia-reperfusion mice, AAV-shFosl1 mice had a reduced infarct size and preserved cardiac function compared with control AAV mice. Our findings suggest a critical role of the FOSL1/MYH9 axis in hindering Ang II-induced vascular remodeling, and we identified FOSL1 as a potential therapeutic target in endothelial cell injuries induced by myocardial ischemia-reperfusion.

Mechanical load regulates the proliferation of multiple cell types in the heart

Abstract Funding Acknowledgements None. Introduction The adult mammalian heart has a poor regenerative and angiogenic potential. At the same time, both primary cardiac tumors and cardiac metastases are extremely rare. This suggests that shared mechanisms may halt the proliferation of cardiac (both cardiomyocyte and endothelial) and cancer cells. Among the mechanisms that have been claimed to be responsible for the loss of proliferative potential of cardiomyocytes early after birth is a sudden increase in mechanical load. Purpose Here, we investigate whether alterations in mechanical load affect the proliferation of multiple cell types in the heart. Methods Adult heart unloading in vivo was achieved using a mouse model of heterotopic heart transplantation, followed by EdU injection to label proliferating cells. Engineered heart tissues (EHTs) were generated using neonatal cardiomyocytes and fibroblasts, with a modified protocol allowing to finely tune mechanical load. Primary cardiac endothelial cells and multiple types of cancer cells (lung, melanoma and colon cancer) along with adult cardiac endothelial cells were included in EHTs. Results In line with our hypothesis, unloaded, heterotransplanted hearts showed a significant number of proliferating cardiomyocytes and endothelial cells, which were almost absent in native hearts. While cancer cells did not grow in native hearts, they massively proliferated in unloaded hearts. Consistent with in vivo results, mechanical unloading in EHTs significantly increased the percentage of EdU-positive cardiomyocytes, endothelial and cancer cells. In contrast, increasing afterload reduced cell proliferation in all cell types. Mechanistically, we identified Nesprin2 as an essential factor in sensing mechanical load and translating it into a cell proliferation arrest. Conclusions Overall, these data indicate that variations in mechanical load have a dramatic effect on the proliferation of multiple cell types within the heart tissue, including cardiomyocytes, endothelial and cancer cells and that Nesprin2 is required to mediate this effect.Unloading in the heartUnloading in EHT

Phosphoproteomic response of cardiac endothelial cells to ischemia and ultrasound

Myocardial infarction and subsequent therapeutic interventions activate numerous intracellular cascades in every constituent cell type of the heart. Endothelial cells produce several protective compounds in response to therapeutic ultrasound, under both normoxic and ischemic conditions. How endothelial cells sense ultrasound and convert it to a beneficial biological response is not known. We adopted a global, unbiased phosphoproteomics approach aimed at understanding how endothelial cells respond to ultrasound. Here, we use primary cardiac endothelial cells to explore the cellular signaling events underlying the response to ischemia-like cellular injury and ultrasound exposure in vitro. Enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. Application of pathway algorithms reveals numerous protein networks recruited in response to ultrasound including those regulating RNA splicing, cell-cell interactions and cytoskeletal organization. Our dataset also permits the informatic prediction of potential kinases responsible for the modifications detected. Taken together, our findings begin to reveal the endothelial proteomic response to ultrasound and suggest potential targets for future studies of the protective effects of ultrasound in the ischemic heart.

Damage to cardiac vasculature may be associated with breast cancer treatment-induced cardiotoxicity

BackgroundBreast cancer is the most common female cancer worldwide. Effective therapies including doxorubicin and trastuzumab have improved survival, but are associated with a substantial risk of cardiovascular disease. Mechanisms underlying cancer treatment-induced cardiotoxicity (CTC) are poorly understood and have largely focused on cardiomyocyte damage, although other cellular populations in the heart such as the cardiac endothelium, may play an important role in cardiac damage. We treated a breast tumor-bearing mouse model with doxorubicin and trastuzumab to investigate the role of the cardiac endothelium in the development of CTC.MethodsImmune compromised mice were inoculated in the 4th mammary fat pad with human breast cancer cells overexpressing HER2 (BT474). When tumors were palpable, mice were treated weekly with doxorubicin (5 mg/kg) and trastuzumab (4 mg/kg). The cardiac phenotype of mice was assessed by echocardiography and histological evaluation of the heart. Cardiac vascular damage was assayed by in vivo permeability assays and primary cultures of murine cardiac endothelial cells were used to assay doxorubicin toxicity in vitro.ResultsThe growth of BT474 breast tumors in Balb/c Nude mice was suppressed upon treatment with doxorubicin and trastuzumab. Mice treated for 4 months with doxorubicin and trastuzumab maintained body weights, but demonstrated an echocardiographic phenotype consistent with preserved left ventricular (LV) ejection fraction, decreased LV mass and increased filling pressures (E/e’). Histological staining with Masson’s trichrome and Picrosirius red showed extensive fibrosis and increased collagen deposition in the ventricular myocardium surrounding blood vessels of treated mice compared to untreated mice. Evans blue permeability assays demonstrated increased cardiac vasculature permeability while primary cardiac endothelial cells exposed to doxorubicin in vitro showed increased cell death as compared to lung or liver endothelial cells.ConclusionsAn orthotopic mouse model of human breast cancer in Nude mice treated with doxorubicin and trastuzumab resulted in a cardiac vascular defect accompanied by preserved LV ejection fraction, decreased LV mass, suggesting mild diastolic dysfunction and cardiac remodeling consistent with subclinical cardiotoxicity. Our data suggest that cardiac endothelium is more sensitive to doxorubicin therapy as compared to other organ endothelium and cardiac endothelial damage may correlate with breast cancer treatment-induced cardiotoxicity.

LncRNA LUADT1 sponges miR-195 to prevent cardiac endothelial cell apoptosis in sepsis

BackgroundThe oncogenic role of the newly identified lncRNA LUADT1 has been revealed in lung adenocarcinoma. It was reported that LUADT1 plays a critical role in multiple human diseases. This study was carried out to investigate the role of LUADT1 in sepsis.MethodsSixty patients with sepsis and sixty healthy volunteers were recruited for this study. Plasma samples were collected from all participants. Human primary coronary artery endothelial cells were also used in this study. The expression of Pim-1, miR-195 and LUADT1 were detected by RT-qPCR. The interaction between miR-195 and LUADT1 was determined by overexpression experiments and luciferase activity assay. Cell apoptosis was detected by flow cytometry. The expression of apoptosis-related protein was detected by Western blotting.ResultsBioinformatics analysis revealed the potential interaction between LUADT1 and miR-195, which was confirmed by dual luciferase reporter assay. LUADT1 was downregulated in patients with sepsis. Moreover, LPS treatment downregulated the expression of LUADT1 in primary cardiac endothelial cells. Overexpression of LUADT1 and miR-195 did not affect the expression of each other in primary cardiac endothelial cells. Interestingly, overexpression of LUADT1 was found to upregulate the expression of Pim-1, a target of miR-195. In addition, it was found that overexpression of LUADT1 and Pim-1 reduced the enhancement effects of miR-195 on LPS-induced cardiac endothelial cell apoptosis.ConclusionIn summary, LUADT1 may protect cardiac endothelial cells against apoptosis in sepsis by regulating the miR-195/Pim-1 axis.

Gene expression in immortalized versus primary isolated cardiac endothelial cells

Endothelial cells take pivotal roles in the heart and the vascular system and their differentiation, subspecification and function is determined by gene expression. A stable, in vitro cardiac endothelial cell line could provide high cell numbers as needed for many epigenetic analyses and facilitate the understanding of molecular mechanisms involved in endothelial cell biology. To test their suitability for transcriptomic or epigenetic studies, we compared the transcriptome of cultured immortalized mouse cardiac endothelial cells (MCEC) to primary cardiac endothelial cells (pEC). Whole transcriptome comparison of MCEC and pEC showed a correlation of 0.75–0.77. Interestingly, correlation of gene expression declined in endothelial cell-typical genes. In MCEC, we found a broad downregulation of genes that are highly expressed in pEC, including well-described markers of endothelial cell differentiation. Accordingly, systematic analysis revealed a downregulation of genes associated with typical endothelial cell functions in MCEC, while genes related to mitotic cell cycle were upregulated when compared to pEC. In conclusion, the findings from this study suggest that primary cardiac endothelial cells should preferably be used for genome-wide transcriptome or epigenome studies. The suitability of in vitro cell lines for experiments investigating single genes or signaling pathways should be carefully validated before use.

SU5416 does not attenuate early RV angiogenesis in the murine chronic hypoxia PH model

BackgroundRight ventricular (RV) angiogenesis has been associated with adaptive myocardial remodeling in pulmonary hypertension (PH), though molecular regulators are poorly defined. Endothelial cell VEGFR-2 is considered a “master regulator” of angiogenesis in other models, and the small molecule VEGF receptor tyrosine kinase inhibitor SU5416 is commonly used to generate PH in rodents. We hypothesized that SU5416, through direct effects on cardiac endothelial cell VEGFR-2, would attenuate RV angiogenesis in a murine model of PH.MethodsC57 BL/6 mice were exposed to chronic hypoxia (CH-PH) to generate PH and stimulate RV angiogenesis. SU5416 (20 mg/kg) or vehicle were administered at the start of the CH exposure, and weekly thereafter. Angiogenesis was measured after one week of CH-PH using a combination of unbiased stereological measurements and flow cytometry-based quantification of myocardial endothelial cell proliferation. In complementary experiments, primary cardiac endothelial cells from C57 BL/6 mice were exposed to recombinant VEGF (50 ng/mL) or grown on Matrigel in the presence of SU5416 (5 μM) or vehicle.ResultSU5416 directly inhibited VEGF-mediated ERK phosphorylation, cell proliferation, and Kdr transcription, but not Matrigel tube formation in primary murine cardiac endothelial cells in vitro. SU5416 did not inhibit CH-PH induced RV angiogenesis, endothelial cell proliferation, or RV hypertrophy in vivo, despite significantly altering the expression profile of genes involved in angiogenesis.ConclusionsThese findings demonstrate that SU5416 directly inhibited VEGF-induced proliferation of murine cardiac endothelial cells but does not attenuate CH-PH induced RV angiogenesis or myocardial remodeling in vivo.

Abstract 392: Decreased ARNT Mediates Cardiovascular Endothelial Dysfunction in Diabetic Cardiomyopathy

Background: Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy. Methods and Results: Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT -/- ) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT -/- mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway. Conclusion: Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.

Abstract 5: Novel Tools for Identification and Purification of Cardiomyocytes, Fibroblasts and Endothelial Cells From Neonatal Mouse and Rat Hearts

Although isolation of vital cardiac cells from neonatal hearts is one of the most used experimental model in cardiac research, the manual dissociation of neonatal hearts is a laborious and difficult-to-standardize procedure. To shorten sample processing time and increase reproducibility, we developed a fully automated dissociation process followed by magnetic purification of cardiomyocytes (CMs), cardiac fibroblasts (CFs) and endothelial cells (ECs). Neonatal hearts were dissociated within 1 h resulting in high yields of single cells with excellent viability. Depending on age and species following cell ratios were recovered after dissociation: 50-70% CMs, 30-40% CFs, 10-20% ECs. In order to purify CMs from dissociated neonatal hearts, we defined antibody cocktails enabling non-myocyte removal. Our depletion strategy allowed for the enrichment of CMs from whole hearts or preparations of heart chambers with purities of up to 98%. To enable simultaneous detection of CMs and subtypes, we developed recombinant antibodies against general CM markers like alpha Actinin, Myosin Heavy Chain or cardiac Troponin T as well as CM subtype-specific antibodies against MLC2a and MLC2v distinguishing between atrial and ventricular CMs respectively. Besides, we developed magnetic enrichment strategies for the purification of CFs and ECs. Current purification strategies that are solely based on antibodies against EC surface markers do not result in sufficiently pure cardiac ECs. Therefore, we established a 2-step enrichment protocol allowing for the complete removal of contaminating fibroblasts and leukocytes. Our data indicate that this protocol significantly improves the purity of primary cardiac ECs. Functionality of ECs before and after enrichment was proven by Dil-Ac-LDL endocytosis. Similarly, our magnetic enrichment protocol for cardiac fibroblasts revealed homogenous expression of fibroblast markers like Vimentin and Prolyl-Hydroxylase, but virtually no contaminations with CMs or ECs. In summary, we established an automated protocol for dissociation of neonatal hearts enabling subsequent purification, characterization and cultivation of CMs, CFs and ECs which can readily be used for cell culture assays or to generate heart muscle models.

Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury.

The aim of this study was to evaluate the paracrine activity of human epicardial-derived cells (hEPDCs) to screen for secreted vasoprotective factors and develop therapeutics to treat vascular reperfusion injury. Epicardial cells support cardiac development, repair, and remodelling after injury in part, through paracrine activity. We hypothesized that secreted ligands from hEPDCs would protect vascular integrity after myocardial infarction (MI) with reperfusion. During simulated ischaemia in culture (24-48 h), concentrated hEPDC-conditioned medium (EPI CdM) increased survival of primary cardiac endothelial cells. In a rat MI model, EPI CdM treatment reduced vascular injury in vivo after reperfusion. By phospho-receptor tyrosine kinase (RTK) arrays, ELISA, and neutralizing antibody screens, we identified hepatocyte growth factor (HGF) as a key vasoprotective factor in EPI CdM. Unexpectedly, we observed that some of the HGF in EPI CdM formed complexes with polyclonal IgG. Following reperfusion, preparations of HGF/IgG complexes provided greater vascular protection than free HGF with IgG. HGF/IgG complexes localized to blood vessels in vivo and increased HGF retention time after administration. In subsequent screens, we found that 'related to tyrosine kinase' (RYK) receptor was phosphorylated after exposure of cardiac endothelial cells to HGF/IgG complexes, but not to free HGF with IgG. The enhanced protection conferred by HGF/IgG complexes was lost after antibody blockade of RYK. Notably, the HGF/IgG complex is the first 'ligand' shown to promote phosphorylation of RYK. Early treatment with HGF/IgG complexes after myocardial ischaemia with reperfusion may rescue tissue through vasoprotection conferred by c-Met and RYK signalling.

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology.

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.

Syndecan-4 modulates basic fibroblast growth factor 2 signaling in vivo.

Syndecan-4 is one of the principal heparan sulfate-carrying proteins on the cell surface. Unlike other members of syndecan family, syndecan-4 mediates phosphatidylinositol 4,5-bisphosphate 2 (PIP(2))-dependent PKC-alpha activation, and overexpression of syndecan-4 in vitro results in enhanced FGF2 signaling. The present study was designed to test the functional effect of increased syndecan-4 expression in endothelial cells in transgenic mice. Several transgenic mice lines expressing syndecan-4 cDNA under control of human endothelial nitric oxide (NO) synthase (eNOS) promoter were generated. Exogenous syndecan-4 was mainly expressed in the heart, brain, and lungs. In particular, the heart demonstrated the greatest increase in the ratio of transgenic-to-native syndecan-4 gene expression. Vessels from the eNOS-syndecan-4 mice demonstrated more pronounced vasodilation to FGF2 but not to VEGF-A(165), sodium nitroprusside, and A 23187 compared with wild-type mice. To elucidate the mechanism of this effect, we measured NO release from primary cardiac endothelial cells isolated from transgenic or wild-type adult mice. Cells from the eNOS-syndecan-4 transgenic mice had a significant increase in FGF2- and VEGF-A(165)-induced NO release compared with endothelial cells from the wild-type mice. However, the absolute magnitude of this increase was higher for FGF2 than VEGF-A(165). In conclusion, enhanced syndecan-4 expression in mouse cardiac endothelial cells results in preferential augmentation of FGF2 but not VEGF-A(165)-induced NO release.