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Myocardial infarction is a leading cause of morbidity and mortality. While reperfusion is now standard therapy, pathological remodeling leading to heart failure remains a clinical problem. Cellular senescence has been shown to contribute to disease pathophysiology and treatment with the senolytic navitoclax attenuates inflammation, reduces adverse myocardial remodeling and results in improved functional recovery. However, it remains unclear which senescent cell populations contribute to these processes. To identify whether senescent cardiomyocytes contribute to disease pathophysiology post-myocardial infarction, we established a transgenic model in which p16 (CDKN2A) expression was specifically knocked-out in the cardiomyocyte population. Following myocardial infarction, mice lacking cardiomyocyte p16 expression demonstrated no difference in cardiomyocyte hypertrophy but exhibited improved cardiac function and significantly reduced scar size in comparison to control animals. This data demonstrates that senescent cardiomyocytes participate in pathological myocardial remodeling. Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated inflammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodeling by spreading senescence to other cell-types. Collectively this study presents a novel demonstration that senescent cardiomyocytes are major contributors to myocardial remodeling and dysfunction following a myocardial infarction. Therefore, to maximize the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimize senolytic strategies to target this cell lineage.

Introduction Endoglin (ENG) forms a receptor complex with ALK1 in endothelial cells (ECs) to promote BMP9/10 signalling. Loss of function mutations in either ENG or ALK1 genes lead to the inherited vascular disorder hereditary haemorrhagic telangiectasia (HHT), characterised by arteriovenous malformations (AVMs). However, the vessel-specific role of ENG and ALK1 proteins in protecting against AVMs is unclear. For example, AVMs have been described to initiate in arterioles, whereas ENG is predominantly expressed in venous ECs. To investigate whether ENG has any arterial involvement in protecting against AVM formation, we specifically depleted the Eng gene in venous and capillary endothelium whilst maintaining arterial expression, and investigated how this affected the incidence and location of AVMs in comparison with pan-endothelial Eng knockdown. Methods Using the mouse neonatal retinal model of angiogenesis, we first established the earliest time point at which Apj-Cre-ERT2 activity was present in venous and capillary ECs but absent from arterial ECs. We then compared the incidence of AVMs following pan-endothelial or venous/capillary-specific ENG knockout. Results Activation of Apj-Cre-ERT2 with tamoxifen from postnatal day (P) 5 ensured preservation of arterial ENG protein expression. Specific loss of ENG expression in ECs of veins and capillaries led to retinal AVMs at a similar frequency to pan-endothelial loss of ENG. AVMs occurred in the proximal as well as the distal part of the retina consistent with a defect in vascular remodelling during maturation of the vasculature. Conclusion Expression of ENG is not required in arterial ECs to protect against AVM formation.

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFβ2 and IL1β. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2, Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.

ObjectiveMyocardial infarction leads to a rapid innate immune response that is ultimately required for repair of damaged heart tissue. We therefore examined circulating monocyte dynamics immediately after reperfusion of the culprit coronary vessel in STEMI patients to determine whether this correlated with level of cardiac injury. A mouse model of cardiac ischaemia/reperfusion injury was subsequently used to establish the degree of monocyte margination to the coronary vasculature that could potentially contribute to the drop in circulating monocytes.Approach and ResultsWe retrospectively analysed blood samples from 51 STEMI patients to assess the number of non-classical (NC), classical and intermediate monocytes immediately following primary percutaneous coronary intervention. Classical and intermediate monocytes showed minimal change. On the other hand circulating numbers of NC monocytes fell by approximately 50% at 90 minutes post-reperfusion. This rapid decrease in NC monocytes was greatest in patients with the largest infarct size (pConclusionsRapid depletion of NC monocytes from the circulation in STEMI patients following coronary artery reperfusion correlates with the level of acute cardiac injury and involves rapid margination to the coronary vasculature.Graphical AbstractHighlights3-5 bullet points that summarize the major findings of the study.Circulating non classical monocytes show a rapid fall in STEMI patients within 90 minutes of re-opening the culprit coronary artery.The extent of the drop in non classical monocytes correlates with loss of cardiac function and increased infarct size.A mouse model of cardiac ischaemia and reperfusion shows rapid margination of monocytes to the coronary vasculature

A key component of cardiac ischemia-reperfusion injury (IRI) is the increased generation of reactive oxygen species, leading to enhanced inflammation and tissue dysfunction in patients following intervention for myocardial infarction. In this study we hypothesized that oxidative stress, due to ischemia-reperfusion, induces senescence which contributes to the pathophysiology of cardiac IRI. We demonstrate that IRI induces cellular senescence in both cardiomyocytes and interstitial cell populations and treatment with the senolytic drug navitoclax after ischemia-reperfusion improves left ventricular function, increases myocardial vascularization, and decreases scar size. SWATH-MS based proteomics reveal that biological processes associated with fibrosis and inflammation, that were increased following ischemia-reperfusion, were attenuated upon senescent cell clearance. Furthermore, navitoclax treatment reduced the expression of proinflammatory, profibrotic and anti-angiogenic cytokines, including interferon gamma-induced protein-10, TGF-β3, interleukin-11, interleukin-16 and fractalkine. Our study provides proof-of-concept evidence that cellular senescence contributes to impaired heart function and adverse remodeling following cardiac ischemia-reperfusion. We also establish that post-IRI senescent cells play a considerable role in the inflammatory response. Subsequently, senolytic treatment, at a clinically feasible time point, attenuates multiple components of this response and improves clinically important parameters. Thus, cellular senescence represents a potential novel therapeutic avenue to improve patient outcomes following cardiac ischemia-reperfusion.

Cytomegalovirus (CMV) seropositivity in adults has been linked to increased cardiovascular disease burden. Phenotypically, CMV infection leads to an inflated CD8 T-lymphocyte compartment. We employed a 8-colour flow cytometric protocol to analyse circulating T cells in 597 octogenarians from the same birth cohort together with NT-proBNP measurements and followed all participants over 7 years. We found that, independent of CMV serostatus, a high number of CD27−CD28+ CD8 EMRA T-lymphocytes (TEMRA) protected from all-cause death after adjusting for known risk factors, such as heart failure, frailty or cancer (Hazard ratio 0.66 for highest vs lowest tertile; confidence interval 0.51–0.86). In addition, CD27−CD28+ CD8 EMRA T-lymphocytes protected from both, non-cardiovascular (hazard ratio 0.59) and cardiovascular death (hazard ratio 0.65). In aged mice treated with the senolytic navitoclax, in which we have previously shown a rejuvenated cardiac phenotype, CD8 effector memory cells are decreased, further indicating that alterations in T cell subpopulations are associated with cardiovascular ageing. Future studies are required to show whether targeting immunosenescence will lead to enhanced life- or healthspan.

Supplemental Digital Content is available in the text., Rationale: ENG (endoglin) is a coreceptor for BMP (bone morphogenetic protein) 9/10 and is strongly expressed in endothelial cells. Mutations in ENG lead to the inherited vascular disorder hereditary hemorrhagic telangiectasia characterized by local telangiectases and larger arteriovenous malformations (AVMs); but how ENG functions to regulate the adult vasculature is not understood. Objective: The goal of the work was to determine how ENG maintains vessel caliber in adult life to prevent AVM formation and thereby protect heart function. Methods and Results: Genetic depletion of endothelial Eng in adult mice led to a significant reduction in mean aortic blood pressure. There was no evidence of hemorrhage, anemia, or AVMs in major organs to explain the reduced aortic pressure. However, large AVMs developed in the peripheral vasculature intimately associated with the pelvic cartilaginous symphysis—a noncapsulated cartilage with a naturally high endogenous expression of VEGF (vascular endothelial growth factor). The increased blood flow through these peripheral AVMs explained the drop in aortic blood pressure and led to increased cardiac preload, and high stroke volumes, ultimately resulting in high-output heart failure. Development of pelvic AVMs in this region of high VEGF expression occurred because loss of ENG in endothelial cells leads to increased sensitivity to VEGF and a hyperproliferative response. Development of AVMs and associated progression to high-output heart failure in the absence of endothelial ENG was attenuated by targeting VEGF signaling with an anti-VEGFR2 (VEGF receptor 2) antibody. Conclusions: ENG promotes the normal balance of VEGF signaling in quiescent endothelial cells to maintain vessel caliber—an essential function in conditions of increased VEGF expression such as local hypoxia or inflammation. In the absence of endothelial ENG, increased sensitivity to VEGF drives abnormal endothelial proliferation in local regions of high VEGF expression, leading to AVM formation and a rapid injurious impact on heart function.

Background Endoglin is a co-receptor for TGFbeta/BMP9/10 signalling and ENG mutations lead to the vascular disorder hereditary haemorrhagic telangiectasia type I (HHT). Endoglin is also required for normal vascular development and angiogenesis, but little is known about endoglin's role in quiescent adult vascular endothelium. Purpose The goal of this present study is to determine how endoglin maintains vessel calibre in adult life to prevent AVM formation and thereby protect heart function. Methods To investigate this role, tamoxifen was administered to adult Cdh5(PAC)-CreERT2; Engfl/fl mice to generate endothelial-specific depletion of endoglin (Eng-iKOe). Cardiac magnetic resonance imaging, myography, vascular casting, microsphere injection, immunohistology, qPCR and aortic telemetry were used to evaluate cardiovascular changes after endoglin knockdown. Results Endothelial-specific loss of endoglin leads to an enlarged heart and cardiomyocyte hypertrophy within 5 weeks, progressing to high output heart failure (HOHF). In vivo aortic telemetry revealed significant loss of aortic pressure within a few days of endoglin depletion. Increased cardiac size and reduced cardiac afterload were confirmed by ventricular pressure loop analysis. As HOHF could result from arteriovenous malformations (AVMs), and these are found primarily in mucocutaneous and pulmonary tissues in HHT, we systematically screened for AVMs using microspheres and vascular casting. Although AVMs were absent in the majority of tissues, they were observed in the pelvic region and may account for the rapid increase in cardiac output. The pelvic cartilaginous symphysis is a noncapsulated cartilage with a naturally high endogenous expression of vascular endothelial growth factor (VEGF). Development of pelvic AVMs in this region of high VEGF expression occurred because loss of endoglin in endothelial cells leads to increased sensitivity to VEGF and a hyper-proliferative response. Finally, we found that inhibition of VEGFR2 was protective against AVMs development, enlargement of the heart and dilatation of the ventricles. Conclusion Our results showed the essential role of endoglin in the maintenance of adult cardio-vasculature through crosstalk with the VEGF signalling pathway. Acknowledgement/Funding British Heart Foundation, Cure HHT, The Swedish Research Council, The Cardiovascular Programme at Karolinska Institutet, The Swedish Cancer Society

Introduction Primary percutaneous coronary intervention (PPCI) is the standard care for treatment of acute myocardial infarction, reducing both mortality and morbidity. However, ischaemia/reperfusion (I/R) injury remains an important complication, contributing up to 50% of the final infarct size. Evidence from our clinical studies suggests that lymphocytes expressing the fractalkine receptor (CX3CR1) are associated with microvascular obstruction and hence poorer long-term patient outcomes. This project aims to investigate the role of fractalkine in lymphocyte-mediated myocardial I/R injury and evaluate whether this damage can be reduced by inhibiting fractalkine/receptor (CX3CL1/CX3CR1) interaction. Methods We are using a mouse model of myocardial I/R injury to study lymphocyte infiltration following MI. Multicolour flow cytometry, immunofluorescence staining, qPCR, imaging mass cytometry and magnetic resonance imaging (MRI) are used to evaluate T cell recruitment and cardiac function in a CX3CR1 knockout mouse line. Results CX3CR1 knockout leads to over 30% reduction in immune cell infiltration at the site of cardiac ischaemic injury 2 hours after reperfusion, both in heterozygous (p ≤ 0.01) and knockout (p ≤ 0.001) mice. By 24 hours I/R, infiltration of CD45+ cells in heterozygous and knockout animals returns to, and surpasses, wild type (WT) levels (p ≤ 0.05). Despite the 20% increase in leukocyte infiltration at 24 hours, we see a 2-fold decrease in T cells at the site of I/R injury in the knockout animals, compared to both WT (p ≤ 0.001) and heterozygous mice (p ≤ 0.0001). T cell infiltration in the knockout is also reduced (although not significantly) when heart infiltrates at 24h reperfusion are investigated by an alternative method: flow cytometry analysis of digested cardiac tissue. Furthermore, our studies show a reduction of 30% in the number of CX3CR1-expressing T cells (p ≤ 0.0001) and 50% in total CX3CR1+ cells (p ≤ 0.01) that infiltrate the left ventricle wall in knockout mice, compared to heterozygous animals. Functional studies using cardiac MRI show no difference between heterozygous and KO mice 6 weeks following I/R injury. Conclusion These findings suggest that knockout of CX3CR1 leads to delayed recruitment of leukocytes following cardiac I/R. This results in an altered immune cell infiltrate including reduced number of T cells in the injured tissue. In addition, the role for CX3CR1-expressing T cells in myocardial I/R is supported by our data showing that the number of CX3CR1+ CD3+ cells is significantly reduced from heterozygous to knockout animals following injury. Conflict of interest None

Ageing is the biggest risk factor for impaired cardiovascular health, cardiovascular disease being the leading cause of death in 40% of individuals over 65 years old. Ageing is associated not only with an increased prevalence of cardiovascular disease but also with a poorer prognosis, including increased mortality or incidence of heart failure after myocardial infarction (MI). We have demonstrated that aged (23 month old) mice have an accumulation of cardiomyocyte senescence, reduced regenerative potential and display increased mortality as well as impaired recovery following MI. Cellular senescence is defined not only by the irreversible loss of division potential but also by the production of a senescence-associated secretory phenotype (SASP). This cocktail of pro-inflammatory cytokines, chemokines, matrix proteases and growth factors can impact on tissue function, inducing fibrosis, extracellular matrix degeneration and driving inflammation. We have therefore begun to test if clearance of senescent cardiomyocytes, using the senolytic compound Navitoclax, has the potential to improve cardiac health and post MI outcomes in aged animals. Following treatment with Navitoclax, but prior to MI, aged mice demonstrated a reduction in senescent cardiomyocytes which was associated increased cardiomyocyte generation, a decline in myocardial hypertrophy and a decrease in fibrosis. Following MI, Navitoclax treated mice displayed a tendency towards improved survival and had a significant improvement in cardiac function when compared to vehicle controls. We conclude that clearance of senescent cells is a potential therapeutic strategy for the treatment of age related cardiac dysfunction.

Objectives Endoglin is a co-receptor for TGFbeta/BMP9/10 signalling and ENG mutations lead to the vascular disorder hereditary haemorrhagic telangiectasia type I (HHT). Endoglin is also required for normal vascular development and angiogenesis, but little is known about endoglin’s role in quiescent adult vascular endothelium. Methods To investigate this role, tamoxifen was administered to adult Cdh5(PAC)-CreERT2;Engfl/fl mice to generate endothelial-specific depletion of endoglin (Eng-iKOe). Cardiac magnetic resonance imaging, myography, vascular casting, microsphere injection, immunohistology, qPCR and aortic telemetry were used to evaluate cardiovascular changes after endoglin knockdown. Results Endothelial-specific loss of endoglin leads to an enlarged heart and cardiomyocyte hypertrophy within 5 weeks, progressing to high output heart failure (HOHF). In vivo aortic telemetry revealed significant loss of aortic pressure within a few days of endoglin depletion. Increased cardiac size and reduced cardiac afterload were confirmed by ventricular pressure loop analysis. As HOHF could result from arteriovenous malformations (AVMs), and these are found primarily in mucocutaneous and pulmonary tissues in HHT, we systematically screened for AVMs using microspheres and vascular casting. Although AVMs were absent in the majority of tissues, they were observed in the pelvic region and may account for the rapid increase in cardiac output. Having also observed an increase of VEGF-A protein in tissues from Eng-iKOe mice, we found that inhibition of VEGFR2 was protective against enlargement of the heart and dilatation of the ventricles. Conclusion Our results showed the essential role of endoglin in the maintenance of adult cardiovasculature through crosstalk with the VEGF signalling pathway.

Introduction Myocardial infarction (MI) is a prominent cause of morbidity and mortality globally. While reperfusion via primary percutaneous coronary intervention is the gold-standard therapy, it can lead to the phenomenon ischemia-reperfusion injury (IRI), characterised by progressive remodelling and heart failure. Little is understood regarding to the mechanisms driving IRI, however, increased oxidative stress is known to play a role. Our previous studies demonstrate that during ageing increased oxidative stress drives telomere associated DNA damage foci (TAF) induced cardiomyocyte senescence, which is directly associated with a hypertrophic phenotype. Furthermore senescent cardiomyocytes express a pro-fibrotic profile; in particular an up-regulation of TGF-β expression. We now hypothesise that cardiomyocyte senescence contributes to remodelling subsequent to MI and IRI via similar mechanisms, and as such represents a potential therapeutic target. Methods Young three month old mice underwent 60 min surgical ligation of the left anterior descending coronary artery to mimic MI followed by reperfusion. Histological analysis at numerous time points post-MI for several senescence markers was performed. Results and conclusions In support of our hypothesis, we have observed that within the surviving myocardium, proximal to the infarct region, cardiomyocytes acquire a senescent-like phenotype. This is demonstrated by an increase in senescence markers including SA-β-Gal, p21 and p16, in addition to an accumulation of TAF. As well as this we verified that mice displayed classical pathophysiological aspects related to MI. Using in vitro studies and transgenic mouse models we aim to better understand the biology underlying cardiomyocyte senescence, and to establish the mechanisms underlying senescence contribution to remodelling. Furthermore we aim to ascertain if modulation or clearance of cardiomyocyte senescence promotes regeneration and improves outcome following IRI.

Endoglin protein (also known as CD105) is a co-receptor for members of the transforming growth factor-β (TGFβ) superfamily of ligands, and regulates angiogenesis. Patients carrying mutations in the endoglin gene develop the inherited vascular dysplasia, Hereditary Haemorrhagic Telangiectasia (HHT). In light of the growing clinical interest in endoglin function in cardiovascular disease, we aimed to determine its role by depleting endoglin in a mouse model during adult life and assess effects on cardiac function. Endoglin was depleted in adult Rosa26-Cre-ERT2;Engfl/fl mice using tamoxifen treatment to generate “ubiquitous” endoglin knockout (Eng-iKOu) mice and cardiac magnetic resonance imaging (MRI) was used to evaluate cardiac function at 1, 3, 5 and 12 weeks after endoglin knockdown. Loss of endoglin leads to an enlarged heart and cardiomyocyte hypertrophy within 5 weeks and left ventricular volumes continued to enlarge substantially over subsequent weeks. However, LV ejection fraction was not significantly altered in Eng-iKOu mice compared with controls suggesting that cardiac function was not impaired. To address whether the cardiac remodelling observed in Eng-iKOu mice was due to loss of endoglin in endothelial cells we used a transgenic Cre line (Cdh5Cre-ERT2) to generate endothelial specific depletion of endoglin (Eng-iKOe). These mice showed a similar increase in heart mass and ventricular volumes to the Eng-iKOu mice. To investigate if vasomotor defects are contributing to the major cardiac remodelling, we analysed vasomotor function in the aortas of Eng-iKOe mice. We found an increased contraction response of the aorta to phenylephrine in Eng-iKOe mice compared to controls, suggesting that endoglin is important in controlling the aortic contractile response. These results describe a novel phenotype and highlight the importance of endothelial endoglin in the maintenance of cardiac structure and function.

Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (ENG) or ACVRL1 (also known as ALK1) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. ENG and ACVRL1 encode receptors for the TGFβ superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling in vivo. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity.

Background Pre-clinical and clinical studies have applied cardiosphere derived cells (CDCs) as a source of stem cells to the injured heart, with the aim of rescuing cardiac function following myocardial infarction (MI). These stem cells, cultured from heart tissue biopsies, are usually prepared at atmospheric oxygen levels (20%) prior to delivery. We hypothesised that pre-conditioning in low oxygen (3%) would prepare the transplanted cells for survival in the hypoxic environment of the ischemic heart and could improve their pro-angiogenic potential. Methods and Results We used mouse heart tissue as a source of CDCs and characterized them based on the expression of endothelial and stem cell markers. We also confirmed the cellular response to hypoxia by monitoring HIF-1α protein expression and induction of vascular endothelial growth factor (VEGF) expression. The effect of hypoxia on the expression of several stem cell markers (Sca1, c-Kit and Abcg2) was analysed using qPCR (quantitative PCR) and FACS (fluorescent activated cell sorting). We found that hypoxia treatment resulted in higher levels of the mesenchymal stem cell marker Sca-1 and Abcg2 in CDCs, although there was no significant change in the expression of c-Kit. These results indicate the potential benefit of hypoxia on increasing some aspects of the ‘stemness’ of cardiac progenitor cells, which could improve outcomes following cell transplantation post MI. To evaluate the potential ability of CDCs to promote angiogenesis in vivo, GFP labelled CDCs were mixed with matrigel, and injected as a subcutaneous plug in adult mice. Immunohistochemical analysis of the plug after 14 days revealed that although most CDCs were not retained in the matrigel plugs, there was an increase in angiogenesis, indicated by increased CD31 expression (in comparison to the group which received matrigel only). To investigate whether pre-treatment of CDCs with hypoxia could improve their pro-angiogenic ability, matrigel containing GFP labelled CDCs pre-cultured in sub-physiological O2 were injected subcutaneously into adult mice. Subsequent immunohistochemical analysis showed that culturing CDCs in sub-physiological O2 increased their pro-angiogenic effect compared to CDCs cultured in normoxia. These data suggest that the pro-angiogenic potential of CDCs is likely to be enhanced by preconditioning in sub-physiological O2 and this may be useful for pro-angiogenic therapy following myocardial infarction.

Cardiosphere-derived cells (CDCs) have been shown to promote cardiac repair in vivo, however the mechanisms involved are poorly understood. Endoglin is a pro-angiogenic TGFβ co-receptor highly expressed by CDCs. Endoglin heterozygous human mononuclear cells (MNCs) demonstrate reduced efficacy in promoting cardiac repair in a mouse model. Therefore we hypothesise that endoglin acts as a key functional regulator of CDCs. The aims of this study were to confirm that CDCs significantly improve cardiac function and augment neovascularisation following myocardial infarction (MI) and to test the effect of endoglin depletion. CDCs were derived from Engfl/fl;Rosa26-CreERT2 neonatal mice and were treated with or without 4hydroxy-tamoxifen in vitro to deplete endoglin. Acute MI was induced in adult C57Bl/6 mice by permanent ligation of the left anterior descending coronary artery. Wild-type (WT) or endoglin knock-out (Eng-KO) CDCs were delivered by intramyocardial injection into the infarct border zone. At 28 days cardiac MRI revealed that both WT and Eng-KO CDC recipient groups showed an increase in ejection fraction compared with PBS-treated controls and left ventricular volume indices were also reduced suggesting attenuated adverse remodelling. Injection of WT-CDCs led to an increased angiogenic response, which was significantly reduced when Eng-KO cells were transplanted. Initial data suggests that endoglin expression is dispensable for CDC-mediated augmentation of cardiac function in injured hearts; however it is essential for stimulating an enhanced angiogenic response.

Endoglin, a TGFβ co-receptor, is essential for cardiovascular development. However, endoglin also has an important role in fibrosis in adult life. Endoglin heterozygous mice have been shown to have reduced fibrosis in response to renal injury. They also have significantly reduced cardiac function following myocardial infarction. In rat cardiac fibroblasts, endoglin expression is up regulated following stimulation with angiotensin II and TGFβ, resulting in reduced expression of MMP1 and increased expression of collagen I. These effects are inhibited by an endoglin specific antibody. Using our conditional endoglin knockout mice we sought to investigate the role of endoglin in cardiac healing following myocardial infarction. Adult Eng fl/fl Rosa26-Cre ERT2 or control (Eng fl/fl) mice were treated with intraperitoneal injection of tamoxifen for 5 days to activate Cre ERT2 and deplete endoglin by Cre-lox recombination. Mice then underwent surgical coronary artery ligation or sham operation. Cine cardiac MRI was performed 28 days after injury. Measurement of left ventricular (LV) volumes and myocardial mass were made using ImageJ, and parameters of cardiac function were calculated. We found that LV volumes and mass were significantly increased (p