Sphingosine 1-phosphate (S1P) is a bioactive lipid that circulates in plasma bound to high-density lipoproteins (HDL) and albumin. Circulating S1P levels correlate positively with systolic blood pressure (BP) in hypertension and negatively with severity in septic shock and with left ventricular function in heart disease. In mice, isolated deficiency in HDL-S1P and endothelial cell S1P receptor (R)-1 both trigger hypertension, supporting an essential role for HDL-S1P in endothelial function. Physiological roles of albumin-S1P and myocyte S1PRs in the cardiovascular system remain incompletely defined. We report that mice lacking all circulating S1P pools display hypotension and lack of BP increase with age, which contrasts with HDL-S1P deficiency and suggests an essential role for albumin-S1P in cardiovascular homeostasis. Although cardiac output was preserved in a basal state, left ventricular systolic function and contractile reserve were reduced in the absence of circulating S1P. Cardiac function and BP were partially or fully normalized by transfusion of erythrocytes capable of S1P production. Hypotension was accompanied by reduced peripheral resistance, and albumin-S1P, but not S1P complexed to an HDL-like chaperone, dose-dependently increased vascular resistance in isolated perfused kidneys via S1PR3 and S1PR2. Epistatic analysis supported a critical role for S1PR3 in S1P-dependent BP maintenance and pointed to a distinct origin of the cardiac phenotype. We thus uncover an essential role for circulating S1P in maintaining BP and left ventricular systolic function in mice. Our results also highlight distinct functions for the pools of S1P bound to HDL and to albumin, carrying both diagnostic and therapeutic implications.

G protein-coupled receptors (GPCRs) are key regulators of cardiovascular function that provide targets for the treatment of cardiovascular disease. Sphingosine 1-phosphate (S1P) is an erythrocyte- and platelet-derived lipid mediator with cognate GPCRs on endothelial cells (EC), vascular smooth muscle cells (VSMC) and cardiomyocytes. S1P circulates in plasma bound to apolipoprotein M (ApoM)-containing high-density lipoproteins (HDL) and to albumin. Circulating S1P levels correlate positively with systolic blood pressure in hypertension and negatively with severity in septic shock and with left ventricular (LV) function in coronary heart disease. In mice, impaired S1P binding to HDL or signaling to EC both trigger hypertension, supporting an essential role for HDL-S1P in supporting endothelial function. The roles of albumin-S1P and myocyte S1PRs in cardiovascular homeostasis remain incompletely defined. Contrasting isolated HDL-S1P deficiency, we report that non-selective depletion of circulating S1P pools in mice impairs LV contractile function and induces hypotension and resistance to the spontaneous increase in blood pressure with age. Cardiac output was preserved in naive S1P deficient mice by compensatory LV dilation, but cardiac reserve reduced in a dobutamine stress test. These phenotypes tracked with hematopoietic cell S1P production and were partially or fully reversed by erythrocyte transfusion. Hypotension was accompanied by reduced peripheral resistance, and S1P infusion dose-dependently increased vascular resistance in isolated perfused kidneys from wild-type mice but not mice with compound deficiency in S1PR2&3. Epistatic analysis supported a critical role for S1PR3 in S1P-dependent blood pressure regulation and pointed to a distinct origin of the cardiac phenotype. Although circulating S1P is elevated in hypertensive mice and humans, increasing circulating S1P was not sufficient to induce hypertension in naive mice. These observations suggests that albumin-S1P crosses the endothelium in resistance arteries to gain access to contractile VSMC S1P receptors, and that myocyte S1PR signaling is essential for vascular resistance and blood pressure maintenance in mice. They also highlight the role for plasma chaperones in specifying vascular responses to S1P and the relevance of S1P as a biomarker and potential therapeutic target for blood pressure regulation and heart failure. ### Competing Interest Statement The authors have declared no competing interest.

Myocardial strain and T1 mapping offer precise evaluation of cardiac function and tissue characteristics. The aim of this study is to provide normative myocardial strain and T1 values in mice model along with their changes after myocardial infarction (MI) using a tailored acquisition and analysis protocol. Healthy mice had MRI before and after MI. Imaging was performed to assess cardiac function and left ventricle (LV) strain. A Look-Locker Inversion-Recovery sequence was used for T1 maps reconstruction. Gadolinium doses and timing of image acquisitions were optimized. Radial and circumferential strain measurements were conducted using a custom software based on feature tracking. To address ECG signal interference, adjustments were made for accurate strain calculations. An LV dilation in MI compared to wild-type (WT) with a decrease in LV ejection fraction (p < 0.0001) were reported while stroke volume and cardiac output remained preserved (p > 0.21). Normative strain and T1 mapping data were provided revealing an increase in circumferential strain from base to apex (basal: –14 ± 1%, mid: –15 ± 2%, apical: – 21 ± 3%; p < 0.0001) and notable differences in native and post-contrast T1 values between mid-ventricular and apical LV segments. Circumferential strain differences between MI and WT mice were pronounced, with a significant drop in apical strain (p < 0.0001) in MI. Post-MI mice exhibited higher native T1 and lower post-contrast T1 compared to WT. Our methodological approach not only refines imaging sequences but also offers insights into cardiac function and tissue characteristics in mice. By describing normative values of strain and T1, our findings lay the groundwork for future research, particularly in drug therapy.

Introduction Ischemic heart disease, such as myocardial infarction (MI), is a major cause of death worldwide. Cardiomyocytes loss during MI leads to fibrotic scarring, associated with severe impairment of cardiac function and ultimately congestive heart failure. There is currently a lack of treatment for the development of fibrosis, highlighting the need to identify novel therapeutic targets. The transcription factor Osr1 (odd-skipped related 1), whose expression correlates with that of fibro-adipogenic progenitors during acute skeletal muscle injury, has been identified as a new potential candidate. Osr1+ cells are involved in embryonic heart and muscle development, but these cells are not well characterized in the adult heart. Objective The aim of this project is to determine the expression and localization of Osr1+ cells in the healthy adult heart and during MI. Method Transcriptomic profiling was performed by NanoString in human heart tissue obtained after early (< 15 days) or late MI (> 3 months). To follow the Osr1 expression and localization in the heart, we used a reporter mouse model with a lineage tracing of Osr1+ cells by GFP expression induced by tamoxifen. Males and females were studied before and during MI induced by permanent ligation of the left anterior descending coronary artery. Osr1+/GFP+ cells from these mice were characterized by immunostaining, flow cytometry, and RT-qPCR. Results In the healthy adult mice, Osr1+/GFP+ cells were expressed at low levels in the heart. Moreover, a large proportion of these cells expressed several markers of mesenchymal stem cells and fibroblasts, such as Sca-1, mEFSK4, and CD90.2, and a higher expression level of the stem cell marker CD34. During MI in human heart tissue, OSR1 expression increased in the early post-MI phase, correlated with an increase in the expression of pro-fibrotic markers such as PDGFRα, COL1A, and COL3A. Consistently, we observed an increase in Osr1 expression in mouse hearts from 7 days post-MI, particularly in the ischemia-induced fibrotic zone. Furthermore, isolated Osr1+/GFP+ cells showed a significant reduction of the stem cell marker CD34 from 7 days post-MI, while they expressed more of the fibroblast marker, mEFSK4, suggesting a differentiation of Osr1+/CD34+ stem cells into Osr1+/mEFSK4+ fibroblasts during the development of MI-induced fibrosis. Conclusion Thus, Osr1+ cells represent a novel cardiac cell population with multipotent capacity and exhibit a fibrogenic behavior during MI.

Introduction Ischemic heart disease, including myocardial infarction (MI), is the leading cause of death worldwide. MI is characterized by a loss of cardiomyocytes associated with fibrotic scarring, leading to severe impairment of cardiac function and ultimately congestive heart failure. There is currently a lack of treatment against the development of fibrosis, prompting the identification of new therapeutic targets. Odd-skipped related 1 (Osr1), a transcription factor whose expression indicates fibro-adipogenic progenitors during acute muscle injury has been identified as a new potential candidate. Osr1+ cells are involved during the cardiac and muscle embryo development, but these cells are not well characterized in the adult heart. Objective This project aims to determine the expression and localization of Osr1+ cells in the healthy adult heart and during MI. Method A transcriptomic profiling was performed by NanoString in human heart tissues obtained after early (<15days) or late MI (>3months). To follow the Osr1 expression and localization in the heart, we used a reporter mouse model with a lineage tracing of Osr1+ cells by GFP expression induced by tamoxifen injection. Males and females were studied before and during MI induced by permanent ligation of the left anterior descending coronary artery. We characterized the Osr1+/GFP+ cells from these mice by immunostaining and flow cytometry. Results We observed a significant increase in OSR1 expression in human cardiac tissue in the early post-MI phase, which correlated with an increase in the expression of pro-fibrotic markers such as PDGFRα, COL1A, and COL3A. In basal conditions in the adult mouse heart, a large proportion of Osr1+/GFP+ cells did not express CD45, CD31, and Ter119, hematopoietic, endothelial, and erythrocyte markers, respectively. However, we found a significant co-expression with several markers of mesenchymal stem cells and fibroblasts, such as Sca-1, mEFSK4, and CD90.2, and with an even higher expression level of the stem cell marker, CD34. Consistently, isolated Osr1+/GFP+ cells appear to have multipotent capacity when cultured in vitro. We did not observe a change in the number of Osr1+/GFP+ cells in post-MI mouse hearts but we found a significant reduction in the CD34 stem cell marker in Osr1+/GFP+ cells, while they expressed more of the fibroblast marker, mEFSK4. Conclusion Osr1+ cells represent a novel cardiac cell population with multipotent capacity and fibrogenic behavior during MI.

BACKGROUND:The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown.METHODS:AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs.RESULTS:Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and gamma delta T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of gamma delta T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-beta (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-beta blockade using neutralizing anti-TGF-beta antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals.CONCLUSION:Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing.

Background: Myocardial infarction (MI) induces a repair response that ultimately generates a stable fibrotic scar. Although the scar prevents cardiac rupture, an excessive profibrotic response impairs optimal recovery by promoting the development of noncontractile fibrotic areas. The mechanisms that lead to cardiac fibrosis are diverse and incompletely characterized. We explored whether the expansion of cardiac fibroblasts after MI can be regulated through a paracrine action of cardiac stromal cells. Methods: We performed a bioinformatic secretome analysis of cardiac stromal PW1 + cells isolated from normal and post-MI mouse hearts to identify novel secreted proteins. Functional assays were used to screen secreted proteins that promote fibroblast proliferation. The expressions of candidates were subsequently analyzed in mouse and human hearts and plasmas. The relationship between levels of circulating protein candidates and adverse post-MI cardiac remodeling was examined in a cohort of 80 patients with a first ST-segment–elevation MI and serial cardiac magnetic resonance imaging evaluations. Results: Cardiac stromal PW1 + cells undergo a change in paracrine behavior after MI, and the conditioned media from these cells induced a significant increase in the proliferation of fibroblasts. We identified a total of 12 candidates as secreted proteins overexpressed by cardiac PW1 + cells after MI. Among these factors, GDF3 (growth differentiation factor 3), a member of the TGF-β (transforming growth factor-β) family, was markedly upregulated in the ischemic hearts. Conditioned media specifically enriched with GDF3 induced fibroblast proliferation at a high level by stimulation of activin-receptor–like kinases. In line with the secretory nature of this protein, we next found that GDF3 can be detected in mice and human plasma samples, with a significant increase in the days after MI. In humans, higher GDF3 circulating levels (measured in the plasma at day 4 after MI) were significantly associated with an increased risk of adverse remodeling 6 months after MI (adjusted odds ratio, 1.76 [1.03–3.00]; P =0.037), including lower left ventricular ejection fraction and a higher proportion of akinetic segments. Conclusions: Our findings define a mechanism for the profibrotic action of cardiac stromal cells through secreted cardiokines, such as GDF3, a candidate marker of adverse fibrotic remodeling after MI. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01113268.

Figure S1- Phantom spectra of succinate at 4.7T and 3 T. Figure S2- SUCCES in Sdhb-/- allografted tumors in mice. Figure S3 - SUCCES in SDHx-mutated PPGL. Figure S4 - SUCCES in non SDHx-mutated PPGL.

Therapies for metastatic SDHB-dependent pheochromocytoma and paraganglioma (PPGL) are limited and poorly efficient. New targeted therapies and identification of early non-invasive biomarkers of response are thus urgently needed for these patients. We characterized an in vivo allograft model of spontaneously immortalized murine chromaffin cells (imCC) with inactivation of the Sdhb gene by dynamic contrast-enhanced MRI (DCE-MRI) and 18FDG-PET. We evaluated the response to several therapies: IACS-010759 (mitochondrial respiratory chain complex I inhibitor), sunitinib (tyrosine kinase inhibitor with anti-angiogenic activity), talazoparib (poly ADP ribose polymerase (PARP) inhibitor) combined or not to temozolomide (alkylating agent), pharmacological inhibitors of HIF2a (PT2385 and PT2977 (belzutifan)) and molecular inactivation of HIF2a (imCC Sdhb-/- shHIF2a). Multimodal imaging was performed, including magnetic resonance spectroscopy (1H-MRS) to monitor the level of succinate in vivo. The allografted model of Sdhb-/- imCC reflected SDHB-deficient tumors, with increased angiogenesis and a particular avidity for 18FDG. After 14 days of treatment, IACS-010759, sunitinib and talazoparib at high doses allowed a significant reduction of the tumor volumes. In contrast to the tumor growth inhibition observed in Sdhb-/- shHIF2a imCC tumors, pharmacological inhibitors of HIF2a (PT2385 and belzutifan) showed no antitumor action in this model, alone or in combination with sunitinib. 1H-MRS, but not DCE-MRI, enabled the monitoring response to sunitinib, which was the best treatment in this study, promoting a decrease in succinate levels detected in vivo. This study paves the way for new therapeutic options and reveals a potential new early biomarker of response to treatment in SDHB-dependent PPGL.

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL- mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10–20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80–100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL -related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

Abstract Purpose: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. Whereas most PPGLs are benign, up to 20% may become metastatic with SDHB- and FH-mutated tumors showing the higher risk. We aimed at determining the contribution of immortalization mechanisms to metastatic progression. Experimental Design: Immortalization mechanisms were investigated in 200 tumors. To identify telomerase (+) tumors, we analyzed genomic alterations leading to transcriptional activation of TERT comprising promoter mutations, hypermethylation and gain copy number. To identify tumors that activated the alternative lengthening of telomere (ALT) mechanism, we combined analyses of telomere length by slot blot, telomere heterogeneity by telomere FISH, and ATRX mutations by next-generation sequencing. Univariate/multivariate and metastasis-free survival (MFS) and overall survival (OS) analyses were carried out for assessment of risk factors and clinical outcomes. Results: Only 37 of 200 (18.5%) tumors achieved immortalization. Telomerase activation occurred in 12 metastatic tumors and was prevalent in SDHB-mutated paragangliomas (P = 2.42e−09). ALT features were present in 25 tumors, mostly pheochromocytomas, regardless of metastatic status or molecular group (P = 0.169), yet ATRX mutations were found preferentially in SDHB/FH-mutated metastatic tumors (P = 0.0014). Telomerase activation and ATRX mutations were independent factors of poor prognosis: MFS (hazard ratio, 48.2 and 33.1; P = 6.50E−07 and 1.90E−07, respectively); OS (hazard ratio, 97.4 and 44.1; P = 4.30E−03 and 2.00E−03, respectively) and were associated with worse MFS and OS (log-rank tests P < 0.0001). Conclusions: Assessment of telomerase activation and ATRX mutations could be used to identify metastatic PPGLs, particularly in tumors at high risk of progression.

Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a dismal prognosis. Genomic studies have enabled progress in our understanding of the molecular bases of ACC, but factors that influence its prognosis are lacking. Amplification of the gene encoding the transcription factor steroidogenic factor-1 (SF-1; also known as NR5A1) is one of the genetic alterations common in ACC. We identified a transcriptional regulatory mechanism involving increased abundance of VAV2, a guanine nucleotide exchange factor for small GTPases that control the cytoskeleton, driven by increased expression of the gene encoding SF-1 in ACC. Manipulating SF-1 and VAV2 abundance in cultured ACC cells revealed that VAV2 was a critical factor for SF-1-induced cytoskeletal remodeling and invasion in culture (Matrigel) and in vivo (chicken chorioallantoic membrane) models. Analysis of ACC patient cohorts indicated that greater VAV2 abundance robustly correlated with poor prognosis in ACC patients. Because VAV2 is a druggable target, our findings suggest that blocking VAV2 may be a new therapeutic approach to inhibit metastatic progression in ACC patients.

Abstract Purpose: Germline mutations in genes encoding mitochondrial succinate dehydrogenase (SDH) are found in patients with paragangliomas, pheochromocytomas, gastrointestinal stromal tumors, and renal cancers. SDH inactivation leads to a massive accumulation of succinate, acting as an oncometabolite and which levels, assessed on surgically resected tissue are a highly specific biomarker of SDHx-mutated tumors. The aim of this study was to address the feasibility of detecting succinate in vivo by magnetic resonance spectroscopy. Experimental Design: A pulsed proton magnetic resonance spectroscopy (1H-MRS) sequence was developed, optimized, and applied to image nude mice grafted with Sdhb−/− or wild-type chromaffin cells. The method was then applied to patients with paraganglioma carrying (n = 5) or not (n = 4) an SDHx gene mutation. Following surgery, succinate was measured using gas chromatography/mass spectrometry, and SDH protein expression was assessed by immunohistochemistry in resected tumors. Results: A succinate peak was observed at 2.44 ppm by 1H-MRS in all Sdhb−/−-derived tumors in mice and in all paragangliomas of patients carrying an SDHx gene mutation, but neither in wild-type mouse tumors nor in patients exempt of SDHx mutation. In one patient, 1H-MRS results led to the identification of an unsuspected SDHA gene mutation. In another case, it helped define the pathogenicity of a variant of unknown significance in the SDHB gene. Conclusions: Detection of succinate by 1H-MRS is a highly specific and sensitive hallmark of SDHx mutations. This noninvasive approach is a simple and robust method allowing in vivo detection of the major biomarker of SDHx-mutated tumors. Clin Cancer Res; 22(5); 1120–9. ©2015 AACR.

Introduction: Cardiac commitment of cells and biomimetic scaffolds have been shown to independently improve the therapeutic efficacy of stem cells. Hypothesis: We tested the combination of these two approaches in a rat model of myocardial infarction subjected to a prolonged follow-up. Methods: Eighty rats underwent permanent coronary artery ligation. Five to 7 weeks after myocardial infarction, those with an echocardiographically-measured ejection fraction (EF) ≤ 55% were reoperated on and randomly allocated to receive an epicardial cell-free fibrin patch (n=25), a fibrin patch loaded with 700,000 human embryonic stem cell (ESC)-derived SSEA1+ cardiac progenitor cells (n=30) or to serve as sham-operated controls (n=25). LV function was assessed monthly until 4 months. Hearts were then processed for the assessment of fibrosis, angiogenesis and cell proliferation and the tissue content of multiple cytokines and growth factors (by qPCR). A heart failure score was then built by integrating the absolute change...

BACKGROUND: Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination.METHODS: Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) <= 55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1(+) progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner.RESULTS: The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified.CONCLUSIONS: A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect. (C) 2015 International Society for Heart and Lung Transplantation. All-rights reserved.

Vascular Ehlers–Danlos syndrome is a dramatic inherited disease caused by mutations of type III collagen ( COL3A1 ) gene, associated with early-onset occurrence of arterial ruptures. Col3a1 +/− heterozygous mice, the only vascular Ehlers–Danlos syndrome model available to date, have no spontaneous early vascular phenotype. Our objective was to determine the susceptibility of Col3a1 +/− mice to develop arterial ruptures under high blood pressure (BP) conditions induced by a 4-week infusion of angiotensin II (AngII). AngII (1 μg/kg per minute) significantly and comparably increased systolic BP in Col3a1 +/− and Col3a1 +/+ mice but led to a higher premature mortality rate in Col3a1 +/− mice compared with Col3a1 +/+ mice (73% versus 36%; P =0.03), particularly during the first-week infusion (55% versus 0%). Echocardiography and histological analysis evidenced that early deaths were caused by thoracic aortic ruptures preceded by dissections and associated with low aortic collagen fibrils content. Remarkably, lowering the dose of AngII (0.5 μg/kg per minute) rescued the first-week premature deaths of Col3a1 +/− mice while decreasing the rises in systolic BP ( P =0.05 compared with the high-dose AngII), resulting in similar mortality rates in both groups of mice at the end of the 4-week period (30% versus 50% in Col3a1 +/− and Col3a1 +/+ mice; P =0.30). Finally, norepinephrine infusion (3.9 μg/kg per minute) did not induced significant mortality in both groups, whereas it significantly increased systolic BP, comparably with the high and with the low dose of AngII in Col3a1 +/− mice ( P =0.53 and P =1.00, respectively). Our findings demonstrated the extreme sensitivity of Col3a1 insufficient mice to prematurely develop thoracic aortic ruptures in response to AngII and its associated high levels in BP.