BACKGROUND:Despite recent significant therapeutic progress, cardiovascular diseases (CVD) remain an unmet clinical, economic, and social burden worldwide. Cell-based therapies have been proposed as therapeutic strategies, however, the overall efficacy was modest. OBJECTIVE:We aimed to fully characterize a novel subpopulation of CD90- mesenchymal cells derived from human heart tissue (hCmPC90-) and evaluate its ability to induce cardiac tissue repair and functional recovery. METHODS:We performed a comprehensive phenotypic characterization of the hCmPC90- by flow cytometry and RNA sequencing. A direct comparison of hCmPC90- with previously clinically tested bone marrow- and cardiac-derived cell types, has been conducted both in vitro by means of various assays of angiogenic potency, and in vivo, by testing the ability to ameliorate left ventricular function in a mouse model of acute myocardial infarction (AMI). RESULTS:hCmPC90- showed distinct surface markers and transcriptional phenotype compared with unselected mesenchymal heart cells (hCmPCs) and the positive CD90 counterpart (hCmPC90+). When human hCmPC90-, hCmPC90+, hCmPC, cardiosphere-derived cells (CDCs), and bone marrow-derived CD34+ cells were functionally tested in vitro, hCmPC90- revealed a superior endothelial differentiation ability, higher anti-inflammatory, cardio-protective capacity, and angiocrine activity. Moreover, hCmPC90- showed specific immune-privileged features. When intramyocardially delivered into infarcted mouse hearts, hCmPC90- outperformed three weeks after injection other clinical-grade cell types, as for left ventricular (LV) function and adverse LV remodeling recovery, infarct size reduction, and vascular density augmentation. CONCLUSION:hCmPC90- shows a superior biological potency which deserves clinical exploitation as an advanced therapy medicinal product in the context of refractory ischemic heart disease.

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterized by impaired left ventricular (LV) diastolic function, with normal LV ejection fraction. Aortic valve stenosis can cause an HFpEF-like syndrome by inducing sustained pressure overload (PO) and cardiac remodeling, as cardiomyocyte (CM) hypertrophy and fibrotic matrix deposition. Recently, in vivo studies linked PO maladaptive myocardial changes and DNA damage response (DDR) activation: DDR-persistent activation contributes to mouse CM hypertrophy and inflammation, promoting tissue remodeling, and HF. Despite the wide acknowledgment of the pivotal role of the stromal compartment in the fibrotic response to PO, the possible effects of DDR-persistent activation in cardiac stromal cell (C-MSC) are still unknown. Finally, this novel mechanism was not verified in human samples. This study aims to unravel the effects of PO-induced DDR on human C-MSC phenotypes. Human LV septum samples collected from severe aortic stenosis with HFpEF-like syndrome patients undergoing aortic valve surgery and healthy controls (HCs) were used both for histological tissue analyses and C-MSC isolation. PO-induced mechanical stimuli were simulated in vitro by cyclic unidirectional stretch. Interestingly, HFpEF tissue samples revealed DNA damage both in CM and C-MSC. DDR-activation markers γH2AX, pCHK1, and pCHK2 were expressed at higher levels in HFpEF total tissue than in HC. Primary C-MSC isolated from HFpEF and HC subjects and expanded in vitro confirmed the increased γH2AX and phosphorylated checkpoint protein expression, suggesting a persistent DDR response, in parallel with a higher expression of pro-fibrotic and pro-inflammatory factors respect to HC cells, hinting to a DDR-driven remodeling of HFpEF C-MSC. Pressure overload was simulated in vitro, and persistent activation of the CHK1 axis was induced in response to in vitro mechanical stretching, which also increased C-MSC secreted pro-inflammatory and pro-fibrotic molecules. Finally, fibrosis markers were reverted by the treatment with a CHK1/ATR pathway inhibitor, confirming a cause-effect relationship. In conclusion we demonstrated that, in severe aortic stenosis with HFpEF-like syndrome patients, PO induces DDR-persistent activation not only in CM but also in C-MSC. In C-MSC, DDR activation leads to inflammation and fibrosis, which can be prevented by specific DDR targeting.

Myocardial aging increases the cardiovascular risk in the elderly. The Receptor for Advanced Glycation End-products (RAGE) is involved in age-related disorders. The soluble isoform (sRAGE) acts as a scavenger blocking the membrane-bound receptor activation. This study aims at investigating RAGE contribution to age-related cardiac remodeling. We analyzed the cardiac function of three different age groups of female Rage-/- and C57BL/6N (WT) mice: 2.5- (Young), 12- (Middle-age, MA) and 21-months (Old) old. While aging, Rage-/- mice displayed an increase in left ventricle (LV) dimensions compared to age-matched WT animals, with the main differences observed in the MA groups. Rage-/- mice showed higher fibrosis and a larger number of α-Smooth Muscle Actin (SMA)+ cells with age, along with increased expression of pro-fibrotic Transforming Growth Factor (TGF)-β1 pathway components. RAGE isoforms were undetectable in LV of WT mice, nevertheless, circulating sRAGE declined with aging and inversely associated with LV diastolic dimensions. Human cardiac fibroblasts stimulated with sRAGE exhibited a reduction in proliferation, pro-fibrotic proteins and TGF-beta Receptor 1 (TGFbR1) expression and Smad2-3 activation. Finally, sRAGE administration to MA WT animals reduced cardiac fibrosis. Hence, our work shows that RAGE associates with age-dependent myocardial changes and indicates sRAGE as an inhibitor of cardiac fibroblasts differentiation and age-dependent cardiac fibrosis.

Background Doxorubicin (Dox) is an anti-cancer anthracycline drug that causes double-stranded DNA breaks. It is highly effective against several types of tumours; however, it also has adverse effects on regenerative populations of normal cells, such as human cardiac mesenchymal progenitor cells (hCmPCs), and its clinical use is limited by cardiotoxicity. Another known effect of Dox is nucleolar disruption, which triggers the ubiquitously expressed nucleolar phosphoprotein Nucleophosmin (NPM) to be released from the nucleolus into the cell, where it participates in the orchestration of cellular stress responses. NPM has also been observed in the extracellular space in response to different stress stimuli; however, the mechanism behind this and its functional implications are as yet largely unexplored. The aim of this study was to establish whether Dox could elicit NPM secretion in the extracellular space and to elucidate the mechanism of secretion and the effect of extracellular NPM on hCmPCs. Results We found that following the double-strand break formation in hCmPCs caused by Dox, NPM was rapidly secreted in the extracellular space by an active mechanism, in the absence of either apoptosis or necrosis. Extracellular release of NPM was similarly seen in response to ultraviolet radiation (UV). Furthermore, we observed an increase of NPM levels in the plasma of Dox-treated mice; thus, NPM release also occurred in vivo. The treatment of hCmPCs with extracellular recombinant NPM induced a decrease of cell proliferation and a response mediated through the Toll-like receptor (TLR)4. We demonstrated that NPM binds to TLR4, and via TLR4, and nuclear factor kappa B (NFkB) activation/nuclear translocation, exerts proinflammatory functions by inducing IL-6 and COX-2 gene expression. Finally, we found that in hCmPCs, NPM secretion could be driven by an autophagy-dependent unconventional mechanism that requires TLR4, since TLR4 inhibition dramatically reduced Dox-induced secretion. Conclusions We hypothesise that the extracellular release of NPM could be a general response to DNA damage since it can be elicited by either a chemical agent such as Dox or a physical genotoxic stressor such as UV radiation. Following genotoxic stress, NPM acts similarly to an alarmin in hCmPCs, being rapidly secreted and promoting cell cycle arrest and a TLR4/NFκB-dependent inflammatory response.

Arrhythmogenic cardiomyopathy (ACM) is hallmarked by ventricular fibro‐adipogenic alterations, contributing to cardiac dysfunctions and arrhythmias. Although genetically determined (e.g., PKP2 mutations), ACM phenotypes are highly variable. More data on phenotype modulators, clinical prognosticators, and etiological therapies are awaited. We hypothesized that oxidized low‐density lipoprotein (oxLDL)‐dependent activation of PPARγ, a recognized effector of ACM adipogenesis, contributes to disease pathogenesis. ACM patients showing high plasma concentration of oxLDL display severe clinical phenotypes in terms of fat infiltration, ventricular dysfunction, and major arrhythmic event risk. In ACM patient‐derived cardiac cells, we demonstrated that oxLDLs are major cofactors of adipogenesis. Mechanistically, the increased lipid accumulation is mediated by oxLDL cell internalization through CD36, ultimately resulting in PPARγ upregulation. By boosting oxLDL in a Pkp2 heterozygous knock‐out mice through high‐fat diet feeding, we confirmed in vivo the oxidized lipid dependency of cardiac adipogenesis and right ventricle systolic impairment, which are counteracted by atorvastatin treatment. The modulatory role of oxidized lipids on ACM adipogenesis, demonstrated at cellular, mouse, and patient levels, represents a novel risk stratification tool and a target for ACM pharmacological strategies.

Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.

Background Left ventricle (LV) and right ventricle (RV) are characterized by well-known physiological differences, mainly related to their different embryological origin, hemodynamic environment, function, structure, and cellular composition. Nevertheless, scarce information is available about cellular peculiarities between left and right ventricular chambers in physiological and pathological contexts. Cardiac mesenchymal stromal cells (C-MSC) are key cells affecting many functions of the heart. Differential features that distinguish LV from RV C-MSC are still underappreciated. Aim To analyze the physiological differential amount, function, and transcriptome of human C-MSC in LV versus (vs.) RV. Methods Human cardiac specimens of LV and RV from healthy donors were used for tissue analysis of C-MSC number, and for C-MSC isolation. Paired LV and RV C-MSC were compared as for surface marker expression, cell proliferation/death ratio, migration, differentiation capabilities, and transcriptome profile. Results Histological analysis showed a greater percentage of C-MSC in RV vs. LV tissue. Moreover, a higher C-MSC amount was obtained from RV than from LV after isolation procedures. LV and RV C-MSC are characterized by a similar proportion of surface markers. Functional studies revealed comparable cell growth curves in cells from both ventricles. Conversely, LV C-MSC displayed a higher apoptosis rate and RV C-MSC were characterized by a higher migration speed and collagen deposition. Consistently, transcriptome analysis showed that genes related to apoptosis regulation or extracellular matrix organization and integrins were over-expressed in LV and RV, respectively. Besides, we revealed additional pathways specifically associated with LV or RV C-MSC, including energy metabolism, inflammatory response, cardiac conduction, and pluripotency. Conclusion Taken together, these results contribute to the functional characterization of RV and LV C-MSC in physiological conditions. This information suggests a possible differential role of the stromal compartment in chamber-specific pathologic scenarios.

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upregulated at early stages of C-MSC adipogenic differentiation and was secreted at higher level over time in ACM- derived C-MSC. Our study provides novel ex vivo and in vitro information on CyPA expression in ACM remodeling paving the way for future C-MSC-based mechanistic and therapeutic investigations.

BACKGROUND:Doxorubicin (DOX) is a chemotherapeutic drug limited in its usefulness by an adverse side effect, cardiotoxicity. The mechanisms leading to this detrimental occurrence are not completely clear, and lately many authors focused their attention on the possible role of microRNAs (miRNAs), small regulators of cardiovascular functions, in this phenomenon. Notably, these molecules recently emerged also as potential circulating biomarkers of several cardiac diseases. Thus, the aim of this study was the simultaneous investigation of circulating and cardiac tissue miRNAs expression upon DOX treatment in vivo. METHODS:Twenty C57BL/6 female mice were administered with 24 mg/Kg cumulative dose of DOX or saline (CTRL) for 2 weeks. Echocardiography was performed at baseline and at the end of treatment (T1). Plasma and heart samples were collected at T1, separating atria from left (LV) and right (RV) ventricles, and miRNAs expression was tested by RT-qPCR-based arrays. All putatively DOX-regulated candidates were then validated by single assays in vivo and then evaluated also in murine immortalized cardiomyocytes (HL-1) treated with 1 μM DOX for 24 h. In the end, bioinformatics target prediction was performed for all DOX-miRNAs. RESULTS:Cardiotoxicity onset was diagnosed upon impairment of six cardiac functional parameters in DOX-treated mice at T1. Samples collection, followed by screening and validation steps, identified eleven miRNAs dysregulated by the drug in plasma, while seven resulted as altered in separate heart chambers. Interestingly, miR-34a-5p and miR-451a showed a dysregulation in both plasma and tissue samples of DOX-administered animals, whereas five additional miRNAs presented chamber specific modulation. Of note, in vitro experiments showed a very modest overlap with in vivo results. Bioinformatics prediction analysis performed on miR-34a-5p and miR-451a identified several putative targets presenting no significant association with cardiotoxicity. Anyhow, the same analyses, conducted by combining all miRNAs regulated by DOX in each heart chamber, evidenced a possible dysregulation of the adherens junctions gene network, known to be involved in the onset and progression of dilated cardiomyopathy, an established detrimental side effect of the drug. CONCLUSIONS:This is the first work investigating miRNAs regulation by DOX both in plasma and heart districts of treated animals. Our results indicate a strong association of miR-34a-5p and miR-451a to DOX-induced cardiotoxicity. In addition, the observed altered expression of diverse miRNAs in separated cardiac chambers hints at a specific response to the drug, implying the existence of different players and pathways leading to dysfunction onset.

Background. Cardiotoxicity is a detrimental side effect of the anticancer drug doxorubicin (DOX), characterized by progressive heart dysfunction. Circulating microRNAs (miRNAs) are recognized as potential biomarkers of cardiac disease; thus, we aimed to investigate their association with late cardiotoxicity in an animal model of disease. Methods. Twenty C57BL/6 female mice were administered with 24 mg/kg cumulative dose of DOX or saline during 2 weeks, followed by a recovery period of one month (T42). Echocardiography was performed at baseline and at T42, and plasma samples were collected at T42. The selection of all miRNAs of interest was conducted by literature overview and by screening, followed by RT-qPCR validation. Results. The analysis of cardiac function at T42 evidenced five DOX-treated animals indistinguishable (NoTox) from controls (CTRLs), while four presented heart impairment (Tox). Our analyses identified eight dysfunction-associated plasma miRNAs. In particular, seven miRNAs were found downregulated in comparison to CTRLs, miR-1-3p, miR-122-5p, miR-127-3p, miR-133a-3p, miR-215-5p, miR-455-3-p, and miR-499a-5p. Conversely, miR-34a-5p showed increased levels in Tox plasma samples. Noteworthy, we determined a cluster composed of miR-1-3p, miR-34a-5p, miR-133a-3p, and miR-499a-5p that distinguished with high-accuracy Tox from NoTox mice. Conclusion. This is the first study indicating that, similarly to what is observed in patients, DOX-administered animals present a differential cardiac response to treatment. Moreover, our results indicate the presence of specific plasma miRNAs whose expression reflect the presence of cardiac dysfunction in response to drug-induced injury.

This record contains raw data related to the article: "Fibrosis Rescue Improves Cardiac Function in Dystrophin-Deficient Mice and Duchenne Patient-Specific Cardiomyocytes by Immunoproteasome Modulation". Abstract Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of b-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibitionedependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD.

The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12–21 week (adult) and 28–40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O 2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.

Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4+ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.

The recruitment of bone marrow ( BM )‐derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension ( PH ). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM ‐derived progenitor cells, we induced pulmonary remodelling in rats by two‐week exposure to chronic hypoxia ( CH , 10% oxygen), a trigger of BM ‐derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH . Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH ‐induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH ‐induced increase in c‐kit + cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c‐kit + cells that colocalize with tyrosine kinase receptor 2 ( VEGF ‐R2) and CD 68 (a marker for macrophages), indicating a positive effect on moderating hypoxia‐induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia‐inducible factor ( HIF )‐1α. Furthermore, sildenafil depressed the number of CXCR 4 + cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM ‐derived c‐kit + cells in the pulmonary tissue. The attenuation of the recruitment of BM ‐derived c‐kit + cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.

Introduction and Hypothesis: The short-lived (6 months) turquoise killifish Nothobranchius furzeri (NF) is a novel and convenient model organism for aging studies in which the impact of microRNAs (...

BACKGROUND:Critical limb ischemia (CLI) is a major health problem with no adequate treatment. Since CLI is characterized by insufficient tissue vascularization, efforts have focused on the discovery of novel angiogenic factors. Cyclophilin A (CyPA) is an immunophilin that has been shown to promote angiogenesis in vitro and to enhance bone marrow (BM) cell mobilization in vivo. However, its potential as an angiogenic factor in CLI is still unknown. Thus, this study aimed to evaluate whether CyPA might induce neo-angiogenesis in ischemic tissues.METHODS AND RESULTS:Wild-type C57Bl/6j mice underwent acute hind-limb ischemia (HLI) and received a single intramuscular administration of recombinant CyPA or saline. Limb perfusion, capillary density and arteriole number in adductor muscles were significantly increased after CyPA treatment. Interestingly, BM-derived CD117(+) cell recruitment was significantly higher in ischemic adductor tissue of mice treated with CyPA versus saline. Therefore, the effect of CyPA on isolated BM-derived CD117(+) cells in vitro was evaluated. Low concentrations of CyPA stimulated CD117(+) cell proliferation while high concentrations promoted cell death. Moreover, CyPA enhanced CD117(+) cell adhesion and migration in a dose-dependent manner. Mechanistic studies revealed that CyPA up-regulated CXCR4 in CD117(+) cells and in adductor muscles after ischemia. Additionally, SDF-1/CXCR4 axis inhibition by the CXCR4 antagonist AMD3100 decreased CyPA-mediated CD117(+) cell recruitment in the ischemic limb.CONCLUSION:CyPA induces neo-angiogenesis by recruiting BM-derived CD117(+) cell into ischemic tissues, at least in part, through SDF-1/CXCR4 axis.

Arterial aging is a major risk factor for the occurrence of cardiovascular diseases. The aged artery is characterized by endothelial dysfunction and vascular smooth muscle cells altered physiology together with low-grade chronic inflammation. MicroRNA-34a (miR-34a) has been recently implicated in cardiac, endothelial, and endothelial progenitor cell senescence; however, its contribution to aging-associated vascular smooth muscle cells phenotype has not been explored so far. We found that miR-34a was highly expressed in aortas isolated from old mice. Moreover, its well-known target, the longevity-associated protein SIRT1, was significantly downregulated during aging in both endothelial cells and vascular smooth muscle cells. Increased miR-34a as well as decreased SIRT1 expression was also observed in replicative-senescent human aortic smooth muscle cells. miR-34a overexpression in proliferative human aortic smooth muscle cells caused cell cycle arrest along with enhanced p21 protein levels and evidence of cell senescence. Furthermore, miR-34a ectopic expression induced pro-inflammatory senescence-associated secretory phenotype molecules. Finally, SIRT1 protein significantly decreased upon miR-34a overexpression and restoration of its levels rescued miR-34a-dependent human aortic smooth muscle cells senescence, but not senescence-associated secretory phenotype factors upregulation. Taken together, our findings suggest that aging-associated increase of miR-34a expression levels, by promoting vascular smooth muscle cells senescence and inflammation through SIRT1 downregulation and senescence-associated secretory phenotype factors induction, respectively, may lead to arterial dysfunctions.