Single-cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia.

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identify CD271+ progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271+ progenitors demonstrate robust in vivo angiogenic capacity over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271+ progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271+ progenitors are strikingly reduced in insulin-resistant donors. Our study highlights the identification of AT-CD271+ progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy.

Single cell transcriptomics identifies adipose tissue CD271+ progenitors for enhanced angiogenesis in limb ischemia.

Therapeutic angiogenesis using mesenchymal stem/stromal cell grafts have shown modest and controversial effects in preventing amputation for patients with critical limb ischemia. Through single-cell transcriptomic analysis of human tissues, we identified CD271 + progenitors specifically from subcutaneous adipose tissue (AT) as having the most prominent pro-angiogenic gene profile distinct from other stem cell populations. AT-CD271 + progenitors demonstrated robust in vivo angiogenic capacity, over conventional adipose stromal cell grafts, characterized by long-term engraftment, augmented tissue regeneration, and significant recovery of blood flow in a xenograft model of limb ischemia. Mechanistically, the angiogenic capacity of CD271 + progenitors is dependent on functional CD271 and mTOR signaling. Notably, the number and angiogenic capacity of CD271 + progenitors was strikingly reduced in insulin resistant donors. Our study highlights the identification of AT-CD271 + progenitors with in vivo superior efficacy for limb ischemia. Furthermore, we showcase comprehensive single-cell transcriptomics strategies for identification of suitable grafts for cell therapy. HIGHLIGHTS: Adipose tissue stromal cells have a distinct angiogenic gene profile among human cell sources. CD271 + progenitors in adipose tissue have a prominent angiogenic gene profile. CD271 + progenitors show superior therapeutic capacities for limb ischemia. CD271 + progenitors are reduced and functionally impaired in insulin resistant donors.

Diabetes impairs the angiogenic capacity of human adipose-derived stem cells by reducing the CD271+ subpopulation in adipose tissue.

Type 2 diabetes mellitus is an important risk factor for cardiovascular diseases (CVDs). Therapeutic angiogenesis using adipose-derived stem cells (ADSCs) is attractive for CVD therapy. However, although it would be critical for ADSC application on CVD therapy, whether and how diabetes impairs human ADSC therapeutic potential is still uncertain. In this study, we aimed to investigate the impact of diabetes on the angiogenic potential of ADSCs in patients with CVDs, with special focus on stemness-related genes and cellular alteration of ADSCs. We established cultured ADSCs from diabetic (DM-ADSCs) and non-diabetic patients (nonDM-ADSCs) with CVDs. DM-ADSCs demonstrated limited proliferative capacity and reduced paracrine capacity of VEGF, with lower expression of the stemness gene SOX2. Angiogenic capacity and ADSC engraftment were assessed using xenograft experiments in a hindlimb ischemia model of athymic nude mice. Consistent with the results of in vitro assays, DM-ADSCs did not rescue limb ischemia. In contrast, nonDM-ADSCs induced neovascularization with enhanced engraftment. To elucidate the mechanism underlying these ADSC changes, we compared the surface marker profiles of freshly isolated ADSCs obtained from diabetic and non-diabetic patients by flow cytometry. Among studied subsets, the CD34+CD31-CD271+ subpopulation was reduced in the adipose tissues of diabetic patients. In addition, SOX2 expression and proliferative capacity were considerably reduced in nonDM-ADSCs derived from the stromal vascular fraction (SVF) with depletion of CD271+ cells (p < 0.01). Our observations elucidated that reduced CD271+ subpopulation is critical for the impairment of ADSCs in diabetic patients. Further investigations on the CD271+ subset of ADSCs might provide novel insights into the mechanisms and solutions for diabetes-related ADSC dysfunction in cell therapy.

Long-Term Administration of Eicosapentaenoic Acid Improves Post-Myocardial Infarction Cardiac Remodeling in Mice by Regulating Macrophage Polarization.

BACKGROUND: Consumption of n-3 fatty acids reduces the incidence of cardiovascular mortality in populations that consume diets rich in fish oil. Eicosapentaenoic acid (EPA) is an n-3 fatty acid known to reduce the frequency of nonfatal coronary events; however, the frequency of mortality after myocardial infarction (MI) is not reduced. The aims of this study were to determine whether long-term administration of EPA regulated cardiac remodeling after MI and to elucidate the underlying therapeutic mechanisms of EPA. METHODS AND RESULTS: C57BL/6J mice were divided into control (phosphate-buffered saline-treated) and EPA-treated groups. After 28 days of treatment, the mice were subjected to either sham surgery or MI by left anterior descending coronary artery ligation. Mortality due to MI or heart failure was significantly lower in the EPA-treated mice than in the phosphate-buffered saline-treated mice. However, the incidence of cardiac rupture was comparable between the EPA-treated mice and the phosphate-buffered saline-treated mice after MI. Echocardiographic tests indicated that EPA treatment attenuated post-MI cardiac remodeling by preventing issues such as left ventricular systolic dysfunction and left ventricle dilatation 28 days after MI induction. Moreover, during the chronic remodeling phase, ie, 28 days after MI, flow cytometry demonstrated that EPA treatment significantly inhibited polarization toward proinflammatory M1 macrophages, but not anti-inflammatory M2 macrophages, in the infarcted heart. Furthermore, EPA treatment attenuated fibrosis in the noninfarcted remote areas during the chronic phase. CONCLUSIONS: Long-term administration of EPA improved the prognosis of and attenuated chronic cardiac remodeling after MI by modulating the activation of proinflammatory M1 macrophages.

Sodium 4-Phenylbutyrate Attenuates Myocardial Reperfusion Injury by Reducing the Unfolded Protein Response.

BACKGROUND: The unfolded protein response (UPR) plays a pivotal role in ischemia-reperfusion (I/R) injury in various organs such as heart, brain, and liver. Sodium 4-phenylbutyrate (PBA) reportedly acts as a chemical chaperone that reduces UPR. In the present study, we evaluated the effect of PBA on reducing the UPR and protecting against myocardial I/R injury in mice. METHODS: Male C57BL/6 mice were subjected to 30-minute myocardial I/R, and were treated with phosphate-buffered saline (as a vehicle) or PBA. RESULTS: At 4 hours after reperfusion, mice treated with PBA had reduced serum cardiac troponin I levels and numbers of apoptotic cells in left ventricles (LVs) in myocardial I/R. Infarct size had also reduced in mice treated with PBA at 48 hours after reperfusion. At 2 hours after reperfusion, UPR markers, including eukaryotic initiation of the factor 2α-subunit, activating transcription factor-6, inositol-requiring enzyme-1, glucose-regulated protein 78, CCAAT/enhancer-binding protein (C/EBP) homologous protein, and caspase-12, were significantly increased in mice treated with vehicle compared to sham-operated mice. Administration of PBA significantly reduced the I/R-induced increases of these markers. Cardiac function and dimensions were assessed at 21 days after I/R. Sodium 4-phenylbutyrate dedicated to the improvement of cardiac parameters deterioration including LV end-diastolic diameter and LV fractional shortening. Consistently, PBA reduced messenger RNA expression levels of cardiac remodeling markers such as collagen type 1α1, brain natriuretic peptide, and α skeletal muscle actin in LV at 21 days after I/R. CONCLUSION: Unfolded protein response mediates myocardial I/R injury. Administration of PBA reduces the UPR, apoptosis, infarct size, and preserved cardiac function. Hence, PBA may be a therapeutic option to attenuate myocardial I/R injury in clinical practice.

Adipose-derived regenerative cells exert beneficial effects on systemic responses following myocardial ischemia/reperfusion.

BACKGROUND: Acute coronary syndrome leads to systemic responses, including activation of the sympathetic nervous system, inflammation of atherosclerotic lesions, changes in metabolism and gene expressions of remote organs such as the spleen, bone marrow, and liver. Clinical trials and experimental studies have demonstrated that therapy with adipose-derived regenerative cells (ADRCs) attenuates myocardial ischemia/reperfusion (I/R) injury. The aim of this study is to investigate the role of ADRCs in regulating systemic reactions following I/R. METHODS: Isolated ADRCs were obtained from green fluorescent protein transgenic male mice. Flow cytometry revealed that freshly isolated ADRCs expressed stem cell markers CD90 and Sca-1, and mesenchymal lineage marker. These cells exhibited multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages. Wild-type mice were subjected to 30 min of left ascending coronary ischemia and 24 h reperfusion. Freshly isolated ADRCs (105 cells) or vehicle (VEH), were administered intravenously through the tail at the time of reperfusion. RESULTS: Compared to VEH, administration of ADRCs significantly reduced circulating troponin levels 24 h after I/R. Using quantitative real-time polymerase chain reaction analysis, the present study confirms that I/R-induced increase of factor X mRNA expression in the liver and was significantly inhibited by ADRCs compared to VEH. Administration of ADRCs significantly reduced the I/R-induced increase in serum levels of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-18 seen in mice receiving VEH. CONCLUSIONS: These results suggest that administration of ADRCs could have an important role in reducing myocardial injury and regulating the hepatic gene expression profile following I/R.

Rho-kinase activation in leukocytes plays a pivotal role in myocardial ischemia/reperfusion injury.

The Rho/Rho-kinase pathway plays an important role in many cardiovascular diseases such as hypertension, atherosclerosis, heart failure, and myocardial infarction. Although previous studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury and cytokine production, the role of Rho-kinase in leukocytes during I/R injury is not well understood. Mice were subjected to 30-min ischemia and reperfusion. Rho-kinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumor necrosis factor (TNF)-α, in leukocytes, compared with saline as the vehicle. Furthermore, fasudil decreased I/R-induced myocardial infarction/area at risk (IA) and I/R-induced leukocyte infiltration in the myocardium. Interestingly, IA in fasudil-administered mice with leukocyte depletion was similar to that in fasudil-administered mice. I/R also resulted in remarkable increases in the mRNA expression levels of the proinflammatory cytokines TNF-α, IL-6, and CCL2 in the heart. Inhibition of Rho-kinase activation in leukocytes has an important role in fasudil-induced cardioprotective effects. Hence, inhibition of Rho-kinase may be an additional therapeutic intervention for the treatment of acute coronary syndrome.

Altered hepatic gene expression profiles associated with myocardial ischemia.

BACKGROUND: Acute coronary syndrome is sometimes accompanied by accelerated coagulability, lipid metabolism, and inflammatory responses, which are not attributable to the cardiac events alone. We hypothesized that the liver plays a pivotal role in the pathophysiology of acute coronary syndrome. We simultaneously analyzed the gene expression profiles of the liver and heart during acute myocardial ischemia in mice. METHODS AND RESULTS: -Mice were divided into 3 treatment groups: sham operation, ischemia/reperfusion, and myocardial infarction. Mice with liver ischemia/reperfusion were included as additional controls. Marked changes in hepatic gene expression were observed after 24 hours, despite the lack of histological changes in the liver. Genes related to tissue remodeling, adhesion molecules, and morphogenesis were significantly upregulated in the livers of mice with myocardial ischemia/reperfusion or infarction but not in those with liver ischemia/reperfusion. Myocardial ischemia, but not changes in the hemodynamic state, was postulated to significantly alter hepatic gene expression. Moreover, detailed analysis of the signaling pathway suggested the presence of humoral factors that intervened between the heart and liver. To address these points, we used isolated primary hepatocytes and showed that osteopontin released from the heart actually altered the signaling pathways of primary hepatocytes to those observed in the livers of mice under myocardial ischemia. Moreover, osteopontin stimulated primary hepatocytes to secrete vascular endothelial growth factor-A, which is important for tissue remodeling. CONCLUSIONS: Hepatic gene expression is potentially regulated by cardiac humoral factors under myocardial ischemia. These results provide new insights into the pathophysiology of acute coronary syndrome.

Altered firing pattern of single-unit muscle sympathetic nerve activity during handgrip exercise in chronic heart failure.

Sympathetic activation in chronic heart failure (CHF) is greatly augmented at rest but the response to exercise remains controversial. We previously demonstrated that single-unit muscle sympathetic nerve activity (MSNA) provides a more detailed description of the sympathetic response to physiological stress than multi-unit nerve recordings. The purpose of this study was to determine whether the reflex response and discharge properties of single-unit MSNA are altered during handgrip exercise (HG, 30% of maximum voluntary contraction for 3 min) in CHF patients (New York Heart Association functional class II or III, n = 16) compared with age-matched healthy control subjects (n = 13). At rest, both single-unit and multi-unit indices of sympathetic outflow were augmented in CHF compared with controls (P < 0.05). However, the percentage of cardiac intervals that contained one, two, three or four single-unit spikes were not different between the groups. Compared to the control group, HG elicited a larger increase in multi-unit total MSNA (Delta1002 +/- 50 compared with Delta636 +/- 76 units min(-1), P < 0.05) and single-unit MSNA spike incidence (Delta27 +/- 5 compared with Delta8 +/- 2 spikes (100 heart beats)(-1)), P < 0.01) in the CHF patients. More importantly, the percentage of cardiac intervals that contained two or three single-unit spikes was increased (P < 0.05) during exercise in the CHF group only (Delta8 +/- 2% and Delta5 +/- 1% for two and three spikes, respectively). These results suggest that the larger multi-unit total MSNA response observed during HG in CHF is brought about in part by an increase in the probability of multiple firing of single-unit sympathetic neurones.

Altered interaction between plasminogen activator inhibitor type 1 activity and sympathetic nerve activity with aging.

BACKGROUND: It has been reported that sympathetic nerve activity (SNA) is associated with fibrinolysis, but the interaction between SNA and the fibrinolytic system with aging has not been elucidated in humans. The purpose of this study was to examine the effect of age-related SNA on the activity of plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (tPA) using muscle SNA (MSNA). METHODS AND RESULTS: This study included 16 young subjects (mean age 26.1 years) and 10 aged subjects (mean age 56.9 years). Lower body negative pressure (LBNP) was performed at -40 mmHg for 30 min. LBNP significantly increased both tPA and PAI-1 activity (from 5.2+/-0.5 to 7.3+/-1.2 IU/ml and from 2.85+/-0.68 to 4.06+/-0.73 U/ml, p<0.01, respectively) in the aged group. In the young group, tPA activity tended to increase, whereas PAI-1 activity was unchanged. There was a correlation between MSNA and PAI-1 activity in the aged group (r=0.47, p<0.01). CONCLUSIONS: SNA in an aging subject leads to an increase in the activity of PAI-1, which indicates that an altered interaction between SNA and PAI-1 activity contributes to increased cardiovascular events in the elderly population.