Mouse Cardiac Endothelial Cells Research Articles

Endocardial HDAC3 is required for myocardial trabeculation

Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration.

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

Cysteine-rich with EGF-like domains 2 (CRELD2) is an endoplasmic reticulum stress-inducible angiogenic growth factor promoting ischemic heart repair

Tissue repair after myocardial infarction (MI) is guided by autocrine and paracrine-acting proteins. Deciphering these signals and their upstream triggers is essential when considering infarct healing as a therapeutic target. Here we perform a bioinformatic secretome analysis in mouse cardiac endothelial cells and identify cysteine-rich with EGF-like domains 2 (CRELD2), an endoplasmic reticulum stress-inducible protein with poorly characterized function. CRELD2 was abundantly expressed and secreted in the heart after MI in mice and patients. Creld2-deficient mice and wild-type mice treated with a CRELD2-neutralizing antibody showed impaired de novo microvessel formation in the infarct border zone and developed severe postinfarction heart failure. CRELD2 protein therapy, conversely, improved heart function after MI. Exposing human coronary artery endothelial cells to recombinant CRELD2 induced angiogenesis, associated with a distinct phosphoproteome signature. These findings identify CRELD2 as an angiogenic growth factor and unravel a link between endoplasmic reticulum stress and ischemic tissue repair.

Oncostatin M/Oncostatin M Receptor Signal Induces Radiation-Induced Heart Fibrosis by Regulating SMAD4 in Fibroblast

Radiation-induced heart fibrosis (RIHF) is a severe consequence of radiation-induced heart damage (RIHD) leading to impaired cardiac function. The involvement of oncostatin M (OSM) and its receptor (OSMR) in RIHD remains unclear. This study aimed to investigate the specific mechanism of OSM/OSMR in RIHF/RIHD. RNA sequencing was performed on heart tissues from a RIHD mouse model. OSM levels were assessed in serum samples obtained from patients receiving thoracic radiation therapy (RT), as well as in RIHF mouse heart tissues and serum using enzyme-linked immunosorbent assay. Fiber activation was evaluated through costimulation of primary cardiac fibroblasts and NIH3T3 cells with RT and OSM, using Western blotting, immunofluorescence, and quantitative Polymerase Chain Reaction (qPCR). Adeno-associated virus serotype 9-mediated overexpression or silencing of OSM specifically in the heart was performed in vivo to assess cardiac fibrosis levels by transthoracic echocardiography and pathologic examination. The regulatory mechanism of OSM on the transcription level of SMAD4 was further explored in vitro using mass spectrometric analysis, chromatin immunoprecipitation-qPCR, and DNA pull-down. OSM levels were elevated in the serum of patients after thoracic RT as well as in RIHF mouse cardiac endothelial cells and mouse serum. The OSM rate (post-RT/pre-RT) and the heart exposure dose in RT patients showed a positive correlation. Silencing OSMR in RIHF mice reduced fibrosis, while OSMR overexpression increased fibrotic responses. Furthermore, increased OSM promoted histone acetylation (H3K27ac) in the SMAD4 promoter region, influencing SMAD4 transcription and subsequently enhancing fibrotic response. The findings demonstrated that OSM/OSMR signaling promotes SMAD4 transcription in cardiac fibroblasts through H3K27 hyperacetylation, thereby promoting radiation-induced cardiac fibrosis and manifestations of RIHD.

Abstract EC.01: Endocardial Hdac3 Promotes Myocardial Trabeculation Through A MicroRNA Pathway

A key process during early heart development is myocardial trabeculation, which is the formation of bundles of myocardium extending into the chamber. The failure of trabeculation often contributes to many forms of congenital heart defects. Proper trabeculation requires coordination among multiple cell types and signaling communication. For instance, growth signals released from the endocardium are required for trabecular myocardial growth and maturation. However, it is poorly understood how these signaling cascades are initiated and regulated in the endocardium. Recently, our lab demonstrated that epicardial histone deacetylase 3 (HDAC3) is required for myocardial compaction. In this current study, we found that the genetic deletion of Histone deacetylase 3 (Hdac3) in the cardiac endothelial cells in mice resulted in early embryo lethality presenting as a hypotrabeculation cardiac phenotype, suggesting that endocardial Hdac3 deletion disrupted critical signals from the endocardium for trabecular myocardium growth. When cultured with supernatant from Hdac3 knockout (KO) mouse cardiac endothelial cells (MCECs), wild-type (WT) mouse embryonic cardiomyocytes also showed decreased proliferation. Single cell RNA-sequencing identified that transforming growth factor ß3 (Tgfß3) along with their extracellular matrix (ECM) downstream targets was significantly downregulated in Hdac3 deficient endocardial cells. Interestingly, chemical inhibition of deacetylase catalytic activity alone resulted in significant downregulation of Tgfß3 in cultured Hdac3 KO MCECs, suggesting that HDAC3 induces Tgfß3 expression in an enzymatic dependent manner. Mechanistically, we identified that miR-129 was significantly upregulated in Hdac3 KO MCECs and Hdac3 endocardial KO hearts. Overexpression of miR-129 repressed Tgfß3 expression in WT MCECs, whereas knockdown of miR-129 restored Tgfß3 expression in Hdac3 KO MCECs. Our findings reveal a critical signaling pathway in which endocardial HDAC3 promotes trabecular myocardium growth by stimulating Tgfß through repressing miR-129, providing novel insights into elucidating the etiology of congenital heart defects and conceptual strategies to promote myocardial regeneration.

Abstract P2116: Role Of Cardiomyocyte Specific Pellino-1, An E3 Ligase On Therapeutic Angiogenesis And Attenuation Of Ventricular Remodeling In A Murine Myocardial Infarction Model

Ubiquitination plays a vital role in our system in controlling vascular inflammation, cellular protein quality control, and minimizing misfolded protein toxicity. Pellino1 (Peli1), a type of E3 ubiquitin ligase, has emerged as a critical regulator of the innate immune response; however, its role in the repair and regeneration of ischemic myocardium remains to be elucidated. Materials: Cardiomyocytes isolated from Wild type (WT), cardiomyocyte-specific Peli1 overexpressed (AMPEL1Tg/+), and cardiomyocyte specific Peli1 knockout (CP1KO) mice were cultured and exposed to 6 h hypoxia, and the conditioned media (CM) collected. CMs were used to treat mouse cardiac endothelial cells (MCECs) followed by H2O2 treatment. Cells were used for Western blot, Flow cytometry, and scratch analyses. For in vivo myocardial infarction (MI) model, animals were exposed to left anterior descending artery (LAD) ligation. Animals were divided into (a) WT, (b) AMPEL1Tg/+(c) CP1KO and subjected to sham (S) and MI. Results: In vitro study documented faster wound closure and increased expression of angiogenic factors (pFlk-1, Ang-1 and VEGF) with MCECs treated with CM obtained from AMPEL1 compared to the other CM obtained from CP1KO and WT. There was no change in cardiac functions between Sham groups. AMPEL1Tg/+ MI group, showed preserved systolic functions, represented by both EF [n=12-14, p&lt;0.0001] and FS [n=12-14, p&lt;0.0001] compared to WTMI. However, CP1KO MI showed loss of systolic function, as represented by EF [n=13, p=0.03] and FS [n=13, p=0.03] compared to WTMI. The extent of fibrosis is also reduced in AMPEL1MI [n=5, p=0.0031], but increased in CP1KOMI [n=6-7, p&lt;0.0001] compared to WTMI. Capillary n=4-5, p=0.0010] and arteriolar density [n=5, p=0.0005] were increased in AMPEL1MI but reduced in CP1KOMI [n=6, p&lt;0.0001]; [n=5-7, p=0.02] compared to WTMI. Western blot analysis documented increased survival and angiogenic factors (pAKT, VEGF, and Bcl2) in AMPEL1MI compared to CP1KOMI and WTMI. Conclusion: Our study uncovers the role of cardiomyocyte-specific Peli1 as a regulator of repair and regeneration of ischemic myocardium, as evidenced by preserved cardiac function, increased expression of angiogenic factors, and reduction of apoptotic markers in the MI model.

Synthesis of N, N, N-trimethyl chitosan-based nanospheres for the prolonged release of curcumin

It is critical to develop a hydrophilic drug carrier with positive charge on the surface to enhance the bioavailability of curcumin to overcome the tissue barrier, e.g., blood brain barrier. In this paper, a quaternized chitosan derivative, N,N,N-Trimethyl Chitosan (TMC) was produced which is a cationic polysaccharide. Nuclear magnetic resonance (1H-NMR) and Infrared Spectroscopy (FTIR) have been used to verify the synthesis of TMC. A simple nanoemulsion process has been developed to produce TMC-based nanosphere to load curcumin. A high encapsulation efficiency (over 90%) can be observed. The average particle size of nanospheres made of TMC with 2.5 mg/mL and 4.0 mg/ mL is estimated at 555.3±117.7 nm and 771.2±123.2 nm, respectively. The effect of the concentrations of TMC on the release profile has been investigated. It is found that nanospheres made of a higher concentration of TMC, 4.0 mg/mL, could lead to an extended release of curcumin, and the first-order release kinetics can be observed when release time increases from 0 to 265 h. The release kinetics of curcumin loaded in TMC nanospheres is also influenced by pH value. In addition, the cytotoxicity study shows that no toxic effect can be found when cells are treated with synthetic TMC. The relative cell viability of mouse cardiac endothelial cells treated with curcumin loaded TMC nanospheres is higher than that when cells treated with curcumin alone.

STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes.

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Abstract P313: Over-expression Of Serine Threonine Kinase 35 Improves Cardiac Function In Streptozotocin Induced Diabetic Mice

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associatedwith impaired responsiveness of ischemic myocardium to proangiogenic factors, subsequentlyleading to heart failure. STK35, a novel kinase that binds to nuclear actin, has been shown toregulate important cellular functions such as cell migration, proliferation, survival, andangiogenesis. Currently, the contribution of altered STK35 expression in human diseases remainsunexplored. In initial studies, we observed that human cardiac biopsies from diabetic patientsshowed a significant decrease in STK35 expression as compared to non-diabetic control hearts.Intriguingly, in a STZ-induced mouse model of diabetes, i.v . injection of rAAV9-STK35 to expressconstitutive STK35 in heart in FVB/N male mice promoted neovascularization and lowered cardiacfibrosis, leading to improved cardiac function of diabetic heart. Our in vitro studies observed highglucose decreased STK35 expression in mouse cardiac endothelial cells (MCEC), whereasSTK35 overexpression increased MCEC migration and vascular tube formation, and upregulatedMCEC to expression of multiple pro-angiogenic proteins. Taken together, our results demonstratethat cardiac-targeted STK35 gene therapy exerts a marked beneficial action by attenuating bothcardiac remodeling and cardiac function in a mouse model of diabetes mellitus. Mechanistically,the beneficial effect may be attributed, at least partially, to enhanced neovascularization in heart.

Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Directed evolution of AAV accounting for long-term and enhanced transduction of cardiovascular endothelial cells in vivo

Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.

Cardiovascular disease risk factors induce mesenchymal features and senescence in mouse cardiac endothelial cells.

Aging, obesity, hypertension, and physical inactivity are major risk factors for endothelial dysfunction and cardiovascular disease (CVD). We applied fluorescence-activated cell sorting (FACS), RNA sequencing, and bioinformatic methods to investigate the common effects of CVD risk factors in mouse cardiac endothelial cells (ECs). Aging, obesity, and pressure overload all upregulated pathways related to TGF-β signaling and mesenchymal gene expression, inflammation, vascular permeability, oxidative stress, collagen synthesis, and cellular senescence, whereas exercise training attenuated most of the same pathways. We identified collagen chaperone Serpinh1 (also called as Hsp47) to be significantly increased by aging and obesity and repressed by exercise training. Mechanistic studies demonstrated that increased SERPINH1 in human ECs induced mesenchymal properties, while its silencing inhibited collagen deposition. Our data demonstrate that CVD risk factors significantly remodel the transcriptomic landscape of cardiac ECs inducing inflammatory, senescence, and mesenchymal features. SERPINH1 was identified as a potential therapeutic target in ECs.

Abstract 16950: Exosomes Derived From Podoplanin Positive Cells Induce Fibrosis and Inflammation in Healthy Mouse Heart

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derived fromactivated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase. In vivo,PDPN-exo were initially injected into the left ventricle of healthy mouse hearts followed withexosomes boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase ofthe end diastolic and systolic volumes. The fibrotic area was characterized by vessels doublepositive to endothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Inconclusion these data suggest that PDPN-exo alter the biology of mCECs, fibroblast andmonocytes and participate in adverse remodeling after MI; their specific cargo may representa cohort of targets for the treatment of cardiac fibrosis.

Abstract 272: Epigenetic Regulation Involved in Diabetes-induced Impairment in Myocardial Reparative Function of Endothelial Progenitor Cell-derived Exosomes

Myocardial infarction (MI) occurs frequently in patients with diabetes resulting in higher mortality and morbidity than non-diabetic patients. We and others have shown that bone marrow-derived endothelial progenitor cells (EPCs) promote cardiac neovascularization and attenuate ischemic injury in animal models. Moreover, emerging evidence supports that exosomes (Exo) mediate stem cell therapy by carrying cell-specific biological signatures and by inducing signaling via transfer of bioactive molecules to target cells. However, autologous cell-based therapies yielded modest clinical results, suggesting that cellular/Exo reparative function may be compromised on a background of disease such as diabetes. In addition, recent studies suggest epigenetic mechanisms, such as histone methylation for gene silencing, promotes diabetes-induced vascular complication. Therefore, we hypothesized that diabetic EPCs produce exosomes of altered and dysfunctional content which compromise EPC reparative function in ischemic heart disease via epigenetic alterations. We collected EPC-Exo from non-diabetic mice (Lepr db/+ ) and diabetic mice (Lepr db/db ) and examined their effect on tube formation and cardiomyocyte/endothelial cell survival in vitro as well as their reparative effects on permanent and acute ischemia/reperfusion (I/R) myocardial ischemic injuries in vivo . Diabetic EPC-Exo promoted neonatal rat cardiomyocyte cell apoptosis under hypoxic stress and repressed endothelial tube formation and cell survival compared to cells treated with WT EPC-Exo. In vivo studies revealed diabetic EPC-Exo significantly attenuated cardiac function, reduced capillary density, increased fibrosis and infarct size in permanent LAD ligation and I/R MI models. Mechanistically,H3K9Me3 was increased in mouse cardiac endothelial cells treated with diabetic EPC-exo, suggesting inhibition of angiogenic genes. Our results provide evidence that diabetic EPC-derived exosomes lose their cardiac reparative activities. Specific angiogenic genes will be examined by CHIP analysis of H3K9Me3. Reversing EPC-Exo function by manipulating H3K9Me3 expression will augment autologous therapies in regenerative medicine.

Abstract 467: Exosomes Derived From Podoplanin Positive Cells Alter Physiology and Structure of Healthy Mouse Heart

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derivedfrom activated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase andSerum Amyloid A3 protein accumulation/mis-folding. In vivo, PDPN-exo were initially injectedinto the left ventricle of healthy mouse hearts followed with exosomes boosters delivered byretro-orbital vein injection. Treated mice developed an extended epicardial fibrosis andamyloidosis with a subsequent impairment in the contractility and increase of the end diastolicand systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. In conclusionthese data suggest that PDPN-exo alter the biology of mCECs, fibroblast and monocytes andparticipate in adverse remodeling after MI; their specific cargo may represent a cohort oftargets for the treatment of cardiac fibrosis.

MiR-96 and miR-183 differentially regulate neonatal and adult postinfarct neovascularization.

Following myocardial infarction (MI), the adult heart has minimal regenerative potential. Conversely, the neonatal heart can undergo extensive regeneration, and neovascularization capacity was hypothesized to contribute to this difference. Here, we demonstrate the higher angiogenic potential of neonatal compared with adult mouse cardiac endothelial cells (MCECs) in vitro and use this difference to identify candidate microRNAs (miRs) regulating cardiac angiogenesis after MI. miR expression profiling revealed miR-96 and miR-183 upregulation in adult compared with neonatal MCECs. Their overexpression decreased the angiogenic potential of neonatal MCECs in vitro and prevented scar resolution and neovascularization in neonatal mice after MI. Inversely, their inhibition improved the angiogenic potential of adult MCECs, and miR-96/miR-183–KO mice had increased peri-infarct neovascularization. In silico analyses identified anillin (ANLN) as a direct target of miR-96 and miR-183. In agreement, Anln expression declined following their overexpression and increased after their inhibition in vitro. Moreover, ANLN expression inversely correlated with miR-96 expression and age in cardiac ECs of cardiovascular patients. In vivo, ANLN+ vessels were enriched in the peri-infarct area of miR-96/miR-183–KO mice. These findings identify miR-96 and miR-183 as regulators of neovascularization following MI and miR-regulated genes, such as anillin, as potential therapeutic targets for cardiovascular disease.

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.

Serelaxin alleviates cardiac fibrosis through inhibiting endothelial-to-mesenchymal transition via RXFP1.

Rationale: Cardiac fibrosis is an integral constituent of every form of chronic heart disease, and persistence of fibrosis reduces tissue compliance and accelerates the progression to heart failure. Relaxin-2 is a human hormone, which has various physiological functions such as mediating renal vasodilation in pregnancy. Its recombinant form Serelaxin has recently been tested in clinical trials as a therapy for acute heart failure but did not meet its primary endpoints. The aim of this study is to examine whether Serelaxin has an anti-fibrotic effect in the heart and therefore could be beneficial in chronic heart failure.Methods: We utilized two different cardiac fibrosis mouse models (ascending aortic constriction (AAC) and Angiotensin II (ATII) administration via osmotic minipumps) to assess the anti-fibrotic potential of Serelaxin. Histological analysis, immunofluorescence staining and molecular analysis were performed to assess the fibrosis level and indicate endothelial cells which are undergoing EndMT. In vitro TGFβ1-induced endothelial-to-mesenchymal transition (EndMT) assays were performed in human coronary artery endothelial cells and mouse cardiac endothelial cells (MCECs) and were examined using molecular methods. Chromatin immunoprecipitation-qPCR assay was utilized to identify the Serelaxin effect on chromatin remodeling in the Rxfp1 promoter region in MCECs.Results: Our results demonstrate a significant and dose-dependent anti-fibrotic effect of Serelaxin in the heart in both models. We further show that Serelaxin mediates this effect, at least in part, through inhibition of EndMT through the endothelial Relaxin family peptide receptor 1 (RXFP1). We further demonstrate that Serelaxin administration is able to increase its own receptor expression (RXFP1) through epigenetic regulation in form of histone modifications by attenuating TGFβ-pSMAD2/3 signaling in endothelial cells.Conclusions: This study is the first to identify that Serelaxin increases the expression of its own receptor RXFP1 and that this mediates the inhibition of EndMT and cardiac fibrosis, suggesting that Serelaxin may have a beneficial effect as anti-fibrotic therapy in chronic heart failure.

Abstract 513: Exosomes Derived From Podoplanin Positive Cells Induce Fibrosis and Inflammation in Healthy Mouse Heart

Superseding fibrosis is the leading cause of the adverse remodeling after myocardial infarction (MI); inflammation and paracrine signals enhance the fibrosis and ventricular dysfunction and inhibit the favorable repair. It has been reported that cells expressing Podoplanin (PDPN), a platelet aggregation- inducing type I transmembrane glycoprotein, appear around 2 days after MI as a signal of activation. We hypothesized that exosomes derived from these cells may actively affect the biology of fibrosis and inflammation. PDPN+ cells were isolated from hearts of mice 2 days after MI, expanded in a selective media and treated with TNFα, Angiotensin II or the combination of both. Exosomes derived from activated PDPN+ cells were isolated from the conditioned media and used in vitro for the treatment of mouse cardiac endothelial cells (mCECs), mouse embryonal fibroblast (MEF) and monocytes and in vivo for the treatment of healthy mouse hearts. Data from q-PCR showed that stimulated PDPN+ cells derived exosomes reprogramed mCECs to the endothelial lymphatic phenotype enhancing the expression of the major lymphatic lineage markers and upregulated the expression of fibrotic markers suggesting an endothelial-mesenchymal transition. Furthermore, stimulated PDPN+ cells derived exosomes drove the fibroblast to myo-fibroblast phenotype and activated monocytes toward pro-inflammatory lineage with an increased expression of TNFα and IL- 1β. In vivo, stimulated PDPN+ cells derived exosomes were initially injected in to the left ventricle of healthy mouse hearts followed with additional boosters delivered by retro-orbital vein injection. Treated mice developed an extended epicardial fibrosis with a subsequent impairment in the contractility and increase in the end diastolic and systolic volumes. In conclusion stimulated PDPN+ cells derived exosomes may impair the biology of mCECs, fibroblast and monocytes leading to adverse remodeling after MI.

Therapeutic Ultrasound Increases Myocardial Blood Flow in Ischemic Myocardium and Cardiac Endothelial Cells: Results of In Vivo and In Vitro Experiments

Therapeutic ultrasound can reduce infarct size in a model of coronary thrombosis even when sonothrombolysis is ineffective. The aim of this study was to test the hypothesis that ultrasound-induced cardioprotection is mediated by molecules released from the vascular endothelium that increase myocardial blood flow (MBF) and also have direct tissue-salvaging effects. Invivo and invitro experiments were performed using a 1.05-MHz transducer. For the invivo experiments 10 control and 10 ultrasound-treated dogs undergoing occlusion of the left anterior descending coronary artery were studied. MBF was measured using myocardial contrast echocardiography. For the invitro experiments, primary mouse cardiac endothelial cells were exposed to ultrasound at baseline or following oxygen-glucose deprivation and endothelial nitric oxide synthase phosphorylation as well as adenosine and the eicosanoids epoxyeicosatrienoic acids, dihydroxyeicosatrienoic acids, and hydroxyl-eicosatetraenoic acids were measured. Invivo, ultrasound treatment caused higher MBF (20±10 vs 10±8, P=.03) and higher wall thickening (3±3% vs 1±0.4%, P=.01) in the collateral-derived border zone compared with control. Epicardial MBF in the left anterior descending coronary artery bed also tended to be higher (17±17 vs 5±4, P=.05) in ultrasound-treated versus control animals; however, endocardial MBF in this region was similar to that in controls (13±14 vs 14±7). Invitro, phosphorylated endothelial nitric oxide synthase and adenosine increased (by 129±11% and 286±63%, respectively, P<.01) with ultrasound compared with unstimulated cells. Similar results were obtained with epoxyeicosatrienoic acids. After oxygen-glucose deprivation, phosphorylated endothelial nitric oxide synthase decreased and was restored with application of ultrasound. Similar changes were noted with epoxyeicosatrienoic acids. Cell viability decreased with oxygen-glucose deprivation and returned to near baseline with ultrasound. Ultrasound increases MBF in ischemic tissue invivo. This effect is likely mediated by the release of a plethora of coronary vasodilators during ultrasound treatment that also have direct tissue-salvaging effects. Therapeutic ultrasound, therefore, has potential for treatment of acute and chronic myocardial ischemia independent of its effect on thrombolysis.