Hypoxia Inducible Factor-1α Potentiates Jagged 1-Mediated Angiogenesis by Mesenchymal Stem Cell-Derived Exosomes.

Insufficient vessel growth associated with ischemia remains an unresolved issue in vascular medicine. Mesenchymal stem cells (MSCs) have been shown to promote angiogenesis via a mechanism that is potentiated by hypoxia. Overexpression of hypoxia inducible factor (HIF)-1α in MSCs improves their therapeutic potential by inducing angiogenesis in transplanted tissues. Here, we studied the contribution of exosomes released by HIF-1α-overexpressing donor MSCs (HIF-MSC) to angiogenesis by endothelial cells. Exosome secretion was enhanced in HIF-MSC. Omics analysis of miRNAs and proteins incorporated into exosomes pointed to the Notch pathway as a candidate mediator of exosome communication. Interestingly, we found that Jagged1 was the sole Notch ligand packaged into MSC exosomes and was more abundant in HIF-MSC than in MSC controls. The addition of Jagged1-containing exosomes from MSC and HIF-MSC cultures to endothelial cells triggered transcriptional changes in Notch target genes and induced angiogenesis in an in vitro model of capillary-like tube formation, and both processes were stimulated by HIF-1α. Finally, subcutaneous injection of Jagged 1-containing exosomes from MSC and HIF-MSC cultures in the Matrigel plug assay induced angiogenesis in vivo, which was more robust when they were derived from HIF-MSC cultures. All Jagged1-mediated effects could be blocked by prior incubation of exosomes with an anti-Jagged 1 antibody. All together, the results indicate that exosomes derived from MSCs stably overexpressing HIF-1α have an increased angiogenic capacity in part via an increase in the packaging of Jagged1, which could have potential applications for the treatment of ischemia-related disease. Stem Cells 2017;35:1747-1759.

RNA-sequencing analysis reveals new alterations in cardiomyocyte cytoskeletal genes in patients with heart failure.

Changes in cardiomyocyte cytoskeletal components, a crucial scaffold of cellular structure, have been found in heart failure (HF); however, the altered cytoskeletal network remains to be elucidated. This study investigated a new map of cytoskeleton-linked alterations that further explain the cardiomyocyte morphology and contraction disruption in HF. RNA-Sequencing (RNA-Seq) analysis was performed in 29 human LV tissue samples from ischemic cardiomyopathy (ICM; n=13) and dilated cardiomyopathy (DCM, n=10) patients undergoing cardiac transplantation and six healthy donors (control, CNT) and up to 16 ICM, 13 DCM and 7 CNT tissue samples for qRT-PCR. Gene Ontology analysis of RNA-Seq data demonstrated that cytoskeletal processes are altered in HF. We identified 60 differentially expressed cytoskeleton-related genes in ICM and 58 genes in DCM comparing with CNT, hierarchical clustering determined that shared cytoskeletal genes have a similar behavior in both pathologies. We further investigated MYLK4, RHOU, and ANKRD1 cytoskeletal components. qRT-PCR analysis revealed that MYLK4 was downregulated (-2.2-fold; P<0.05) and ANKRD1 was upregulated (2.3-fold; P<0.01) in ICM patients vs CNT. RHOU mRNA levels showed a statistical trend to decrease (-2.9-fold). In DCM vs CNT, MYLK4 (-4.0-fold; P<0.05) and RHOU (-3.9-fold; P<0.05) were downregulated and ANKRD1 (2.5-fold; P<0.05) was upregulated. Accordingly, MYLK4 and ANKRD1 protein levels were decreased and increased, respectively, in both diseases. Furthermore, ANKRD1 and RHOU mRNA levels were related with LV function (P<0.05). In summary, we have found a new map of changes in the ICM and DCM cardiomyocyte cytoskeleton. ANKRD1 and RHOU mRNA levels were related with LV function which emphasizes their relevance in HF. These new cytoskeletal changes may be responsible for altered contraction and cell architecture disruption in HF patients. Moreover, these results improve our knowledge on the role of cytoskeleton in functional and structural alterations in HF.

Metabolic syndrome in heart transplantation: impact on survival and renal function.

The aim of our study was to analyze the early presence of metabolic syndrome (MS) in heart transplant (HTx) patients, and to assess its long-term impact on survival and renal function. From January 2000 to October 2011, 253 consecutive HTx patients who survived more than 90 days were included. MS was diagnosed if patients met revised NCEP-ATP III criteria at HTx or within 3 months post-HTx. The prevalence of MS was 41.9%. Patients with MS had greater overall mortality after a mean follow-up of 1700 ± 979 days (log-rank test, P = 0.020). In the multivariate analysis, and subject to a minimum survival of 90 days, the only independent predictor variables of long-term mortality were the presence of MS (OR, odds ratio 2.087, P = 0.032), and rejection episodes (OR 1.833, P = 0.001). Patients with MS had worse renal function at baseline both in plasma creatinine (1.19 ± 0.44 vs. 1.03 ± 0.29 mg/dl, P = 0.002) and glomerular filtration rate estimated by modified diet in renal disease (73.60 ± 26.76 vs. 87.30 ± 43.55 ml/min/1.73 m(2) , P = 0.005), whereas progressive impairment of renal function was of equal magnitude in both groups. The presence of MS prior to transplant or its development within the first 3 months identified a subgroup at greater risk of mortality and long-term renal dysfunction.

CFD analysis of the HVAD's hemodynamic performance and blood damage with insight into gap clearance.

Mechanical circulatory support using ventricular assist devices has become commonplace in the treatment of patients suffering from advanced stages of heart failure. While blood damage generated by these devices has been evaluated in depth, their hemodynamic performance has been investigated much less. This work presents the analysis of the complete operating map of a left ventricular assist device, in terms of pressure head, power and efficiency. Further investigation into its hemocompatibility is included as well. To achieve these objectives, computational fluid dynamics simulations of a centrifugal blood pump with a wide-blade impeller were performed. Several conditions were considered by varying the rotational speed and volumetric flow rate. Regarding the device's hemocompatibility, blood damage was evaluated by means of the hemolysis index. By relating the hemocompatibility of the device to its hemodynamic performance, the results have demonstrated that the highest hemolysis occurs at low flow rates, corresponding to operating conditions of low efficiency. Both performance and hemocompatibility are affected by the gap clearance. An innovative investigation into the influence of this design parameter has yielded decreased efficiencies and increased hemolysis as the gap clearance is reduced. As a further novelty, pump operating maps were non-dimensionalized to highlight the influence of Reynolds number, which allows their application to any working condition. The pump's operating range places it in the transitional regime between laminar and turbulent, leading to enhanced efficiency for the highest Reynolds number.

Human progenitor cells derived from cardiac adipose tissue ameliorate myocardial infarction in rodents.

Myocardial infarction caused by vascular occlusion results in the formation of nonfunctional fibrous tissue. Cumulative evidence indicates that cell therapy modestly improves cardiac function; thus, novel cell sources with the potential to repair injured tissue are actively sought. Here, we identify and characterize a cell population of cardiac adipose tissue-derived progenitor cells (ATDPCs) from biopsies of human adult cardiac adipose tissue. Cardiac ATDPCs express a mesenchymal stem cell-like marker profile (strongly positive for CD105, CD44, CD166, CD29 and CD90) and have immunosuppressive capacity. Moreover, cardiac ATDPCs have an inherent cardiac-like phenotype and were able to express de novo myocardial and endothelial markers in vitro but not to differentiate into adipocytes. In addition, when cardiac ATDPCs were transplanted into injured myocardium in mouse and rat models of myocardial infarction, the engrafted cells expressed cardiac (troponin I, sarcomeric α-actinin) and endothelial (CD31) markers, vascularization increased, and infarct size was reduced in mice and rats. Moreover, significant differences between control and cell-treated groups were found in fractional shortening and ejection fraction, and the anterior wall remained significantly thicker 30days after cardiac delivery of ATDPCs. Finally, cardiac ATDPCs secreted proangiogenic factors under in vitro hypoxic conditions, suggesting a paracrine effect to promote local vascularization. Our results indicate that the population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) may be valid candidates for future use in cell therapy to regenerate injured myocardium.

Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.

Human dental pulp contains precursor cells termed dental pulp stem cells (DPSC) that show self-renewal and multilineage differentiation and also secrete multiple proangiogenic and antiapoptotic factors. To examine whether these cells could have therapeutic potential in the repair of myocardial infarction (MI), DPSC were infected with a retrovirus encoding the green fluorescent protein (GFP) and expanded ex vivo. Seven days after induction of myocardial infarction by coronary artery ligation, 1.5 x 10(6) GFP-DPSC were injected intramyocardially in nude rats. At 4 weeks, cell-treated animals showed an improvement in cardiac function, observed by percentage changes in anterior wall thickening left ventricular fractional area change, in parallel with a reduction in infarct size. No histologic evidence was seen of GFP+ endothelial cells, smooth muscle cells, or cardiac muscle cells within the infarct. However, angiogenesis was increased relative to control-treated animals. Taken together, these data suggest that DPSC could provide a novel alternative cell population for cardiac repair, at least in the setting of acute MI.

Mesenchymal Stem Cell Migration and Proliferation Are Mediated by Hypoxia-Inducible Factor-1α Upstream of Notch and SUMO Pathways.

Mesenchymal stem cells (MSCs) are effective in treating several pathologies. We and others have demonstrated that hypoxia or hypoxia-inducible factor 1 alpha (HIF-1α) stabilization improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. To further explore the mechanisms induced by HIF-1α in MSCs, we studied its relationship with Notch signaling and observed that overexpression of HIF-1α in MSCs increased protein levels of the Notch ligands Jagged 1-2 and Delta-like (Dll)1, Dll3, and Dll4 and potentiated Notch signaling only when this pathway was activated. Crosstalk between HIF and Notch resulted in Notch-dependent migration and spreading of MSCs, which was abolished by γ-secretase inhibition. However, the HIF-1-induced increase in MSC proliferation was independent of Notch signaling. The ubiquitin family member, small ubiquitin-like modifier (SUMO), has important functions in many cellular processes and increased SUMO1 protein levels have been reported in hypoxia. To investigate the potential involvement of SUMOylation in HIF/Notch crosstalk, we measured general SUMOylation levels and observed increased SUMOylation in HIF-1-expressing MSCs. Moreover, proliferation and migration of MSCs were reduced in the presence of a SUMOylation inhibitor, and this effect was particularly robust in HIF-MSCs. Immunoprecipitation studies demonstrated SUMOylation of the intracellular domain of Notch1 (N1ICD) in HIF-1-expressing MSCs, which contributed to Notch pathway activation and resulted in increased levels of N1ICD nuclear translocation as assessed by subcellular fractionation. SUMOylation of N1ICD was also observed in HEK293T cells with stabilized HIF-1α expression, suggesting that this is a common mechanism in eukaryotic cells. In summary, we describe, for the first time, SUMOylation of N1ICD, which is potentiated by HIF signaling. These phenomena could be relevant for the therapeutic effects of MSCs in hypoxia or under conditions of HIF stabilization.

New Cell Adhesion Molecules in Human Ischemic Cardiomyopathy. PCDHGA3 Implications in Decreased Stroke Volume and Ventricular Dysfunction.

BACKGROUND: Intercalated disks are unique structures in cardiac tissue, in which adherens junctions, desmosomes, and GAP junctions co-localize, thereby facilitating cardiac muscle contraction and function. Protocadherins are involved in these junctions; however, their role in heart physiology is poorly understood. We aimed to analyze the transcriptomic profile of adhesion molecules in patients with ischemic cardiomyopathy (ICM) and relate the changes uncovered with the hemodynamic alterations and functional depression observed in these patients. METHODS AND RESULTS: Twenty-three left ventricular tissue samples from patients diagnosed with ICM (n = 13) undergoing heart transplantation and control donors (CNT, n = 10) were analyzed using RNA sequencing. Forty-two cell adhesion genes involved in cellular junctions were differentially expressed in ICM myocardium. Notably, the levels of protocadherin PCDHGA3 were related with the stroke volume (r = -0.826, P = 0.003), ejection fraction (r = -0.793, P = 0.004) and left ventricular end systolic and diastolic diameters (r = 0.867, P = 0.001; r = 0.781, P = 0.005, respectively). CONCLUSIONS: Our results support the importance of intercalated disks molecular alterations, closely involved in the contractile function, highlighting its crucial significance and showing gene expression changes not previously described. Specifically, altered PCDHGA3 gene expression was strongly associated with reduced stroke volume and ventricular dysfunction in ICM, suggesting a relevant role in hemodynamic perturbations and cardiac performance for this unexplored protocadherin.

RNA sequencing analysis and atrial natriuretic peptide production in patients with dilated and ischemic cardiomyopathy.

BACKGROUND: The atrium is the major site of ANP synthesis, which has been said to increase in heart failure as a result of increased production in the left ventricular (LV) myocardium. This is a key issue related to its diagnostic and prognostic capabilities. We aimed to evaluate protein levels of proANP and ANP and the enzymes that cleave the natriuretic peptides, corin and furin, in the LV tissue of heart transplant patients with dilated (DCM) and ischemic (ICM) cardiomyopathy compared with control donors (CNT). We also evaluate mRNA levels of ANP gene (NPPA) by RNA sequencing in the same tissue. METHODS AND RESULTS: Seventy-three human LV tissue samples from ICM (n=30) and DCM (n=33) patients and CNT (n=10) were analyzed by Western blot and RNA sequencing. Comparing protein levels according to etiology, neither DCM nor ICM showed levels of proANP or ANP different from those of CNT. However, NPPA was increased in both groups compared to CNT (DCM 32 fold, p<0.0001; ICM 10 fold, p<0.0001). Corin (but not furin) was elevated in the ICM group compared to CNT (112 ± 24 vs. 100 ± 7, p<0.05), and its level was inversely related with LV ejection fraction (LVEF) (r=-0.399, p<0.05). CONCLUSIONS: Patients present with elevated levels of NPPA but not of proANP or ANP proteins in LV tissue, which may be due to posttranscripcional regulation of NPPA or different pathways for ANP secretion between the atrium and ventricle. Moreover, there are differences between DCM and ICM in corin levels, indicating that a different molecular mechanism may exist that converge in this syndrome. Further, LV concentration of corin is inversely related to LVEF in ICM.

Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair.

Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor-1 alpha (HIF-1α) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1α overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1α lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1α on MSC biology and suggests the stabilization of HIF-1α as a novel strategy for cellular therapies.

Heart failure entails significant changes in human nucleocytoplasmic transport gene expression.

BACKGROUND: Heart failure (HF) induces alterations in nucleocytoplasmic transport, which is essential to the cardiomyocyte biology. The objective of this study was to analyze the changes in gene expression in human HF, particularly focusing on nucleocytoplasmic transport-related genes. METHODS AND RESULTS: 29 RNA heart samples from dilated cardiomyopathy (DCM, n = 12) and ischemic cardiomyopathy (ICM, n = 12) patients undergoing heart transplantation and control donors (CNT, n = 5) were extracted to perform a microarray profiling using Affymetrix Human Gene® 1.0 ST arrays. We focused on the study of 5 nucleocytoplasmic transport-related genes, since this functional category has not previously been studied in HF. XPO1, GABPB2, and RANBP17 were upregulated, while KALRN was downregulated in both DCM and ICM, and XPO5 only in DCM. Validation of the results by RT-qPCR increasing the total heart samples up to 41 showed a high degree of consistency with microarray results. Moreover, we observed a strong relationship between the XPO1 mRNA and robust left ventricular function parameters in ICM: left ventricular end-systolic (r = 0.81, p<0.0001) and end-diastolic diameters (r = 0.80, p<0.0001), and ejection fraction (r = -0.57, p<0.05). CONCLUSIONS: We show that the expression of nucleocytoplasmic transport-related genes is altered in HF. Furthermore, XPO1 mRNA level is closely related with robust left ventricular function parameters in ICM patients. These changes may help to distinguish DCM and ICM in HF at the level of the transcriptome and provide a base for novel therapeutic approaches.

Differential gene expression of cardiac ion channels in human dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is characterized by idiopathic dilation and systolic contractile dysfunction of the cardiac chambers. The present work aimed to study the alterations in gene expression of ion channels involved in cardiomyocyte function. METHODS AND RESULTS: Microarray profiling using the Affymetrix Human Gene® 1.0 ST array was performed using 17 RNA samples, 12 from DCM patients undergoing cardiac transplantation and 5 control donors (CNT). The analysis focused on 7 cardiac ion channel genes, since this category has not been previously studied in human DCM. SCN2B was upregulated, while KCNJ5, KCNJ8, CLIC2, CLCN3, CACNB2, and CACNA1C were downregulated. The RT-qPCR (21 DCM and 8 CNT samples) validated the gene expression of SCN2B (p < 0.0001), KCNJ5 (p < 0.05), KCNJ8 (p < 0.05), CLIC2 (p < 0.05), and CACNB2 (p < 0.05). Furthermore, we performed an IPA analysis and we found a functional relationship between the different ion channels studied in this work. CONCLUSION: This study shows a differential expression of ion channel genes involved in cardiac contraction in DCM that might partly underlie the changes in left ventricular function observed in these patients. These results could be the basis for new genetic therapeutic approaches.

Heart failure induces significant changes in nuclear pore complex of human cardiomyocytes.

AIMS: The objectives of this study were to analyse the effect of heart failure (HF) on several proteins of nuclear pore complex (NPC) and their relationship with the human ventricular function. METHODS AND RESULTS: A total of 88 human heart samples from ischemic (ICM, n = 52) and dilated (DCM, n = 36) patients undergoing heart transplant and control donors (CNT, n = 9) were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001), Nup160 (88%, p<0.0001) and Nup153 (137%, p = 0.004) than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001), Nup160 (65%, p<0.0001), Nup153 (155%, p = 0.006) and Nup93 (88%, p<0.0001) compared with CNT. However, Nup155 and translocated promoter region (TPR) did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD) and Nup160 (r = -0382, p = 0.004; r = -0.290, p = 0.033; respectively). CONCLUSIONS: This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93) that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.

IL1ß induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB.

Mesenchymal stem cells are often transplanted into inflammatory environments where they are able to survive and modulate host immune responses through a poorly understood mechanism. In this paper we analyzed the responses of MSC to IL-1ß: a representative inflammatory mediator. Microarray analysis of MSC treated with IL-1ß revealed that this cytokine activateds a set of genes related to biological processes such as cell survival, cell migration, cell adhesion, chemokine production, induction of angiogenesis and modulation of the immune response. Further more detailed analysis by real-time PCR and functional assays revealed that IL-1ß mainly increaseds the production of chemokines such as CCL5, CCL20, CXCL1, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11 and CX(3)CL1, interleukins IL-6, IL-8, IL23A, IL32, Toll-like receptors TLR2, TLR4, CLDN1, metalloproteins MMP1 and MMP3, growth factors CSF2 and TNF-α, together with adhesion molecules ICAM1 and ICAM4. Functional analysis of MSC proliferation, migration and adhesion to extracellular matrix components revealed that IL-1ß did not affect proliferation but also served to induce the secretion of trophic factors and adhesion to ECM components such as collagen and laminin. IL-1ß treatment enhanced the ability of MSC to recruit monocytes and granulocytes in vitro. Blockade of NF-κß transcription factor activation with IκB kinase beta (IKKß) shRNA impaired MSC migration, adhesion and leucocyte recruitment, induced by IL-1ß demonstrating that NF-κB pathway is an important downstream regulator of these responses. These findings are relevant to understanding the biological responses of MSC to inflammatory environments.

Cardiac transcription factors driven lineage-specification of adult stem cells.

Differentiation of human bone marrow mesenchymal stem cells (hBMSC) into the cardiac lineage has been assayed using different approaches such as coculture with cardiac or embryonic cells, treatment with factors, or by seeding cells in organotypic cultures. In most cases, differentiation was evaluated in terms of expression of cardiac-specific markers at protein or molecular level, electrophysiological properties, and formation of sarcomers in differentiated cells. As observed in embryonic stem cells and cardiac progenitors, differentiation of MSC towards the cardiac lineage was preceded by translocation of NKX2.5 and GATA4 transcription factors to the nucleus. Here, we induce differentiation of hBMSC towards the cardiac lineage using coculture with neonatal rat cardiomyocytes. Although important ultrastructural changes occurred during the course of differentiation, sarcomerogenesis was not fully achieved even after long periods of time. Nevertheless, we show that the main cardiac markers, NKX2.5 and GATA4, translocate to the nucleus in a process characteristic of cardiac specification.