Hypoxia-induced miR-210 modulates the inflammatory response and fibrosis upon acute ischemia.

Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-ß1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-ß type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis.

MicroRNA-21/PDCD4 Proapoptotic Signaling From Circulating CD34+ Cells to Vascular Endothelial Cells: A Potential Contributor to Adverse Cardiovascular Outcomes in Patients With Critical Limb Ischemia.

OBJECTIVE: In patients with type 2 diabetes (T2D) and critical limb ischemia (CLI), migration of circulating CD34+ cells predicted cardiovascular mortality at 18 months after revascularization. This study aimed to provide long-term validation and mechanistic understanding of the biomarker. RESEARCH DESIGN AND METHODS: The association between CD34+ cell migration and cardiovascular mortality was reassessed at 6 years after revascularization. In a new series of T2D-CLI and control subjects, immuno-sorted bone marrow CD34+ cells were profiled for miRNA expression and assessed for apoptosis and angiogenesis activity. The differentially regulated miRNA-21 and its proapoptotic target, PDCD4, were titrated to verify their contribution in transferring damaging signals from CD34+ cells to endothelial cells. RESULTS: Multivariable regression analysis confirmed that CD34+ cell migration forecasts long-term cardiovascular mortality. CD34+ cells from T2D-CLI patients were more apoptotic and less proangiogenic than those from control subjects and featured miRNA-21 downregulation, modulation of several long noncoding RNAs acting as miRNA-21 sponges, and upregulation of the miRNA-21 proapoptotic target PDCD4. Silencing miR-21 in control CD34+ cells phenocopied the T2D-CLI cell behavior. In coculture, T2D-CLI CD34+ cells imprinted naive endothelial cells, increasing apoptosis, reducing network formation, and modulating the TUG1 sponge/miRNA-21/PDCD4 axis. Silencing PDCD4 or scavenging reactive oxygen species protected endothelial cells from the negative influence of T2D-CLI CD34+ cells. CONCLUSIONS: Migration of CD34+ cells predicts long-term cardiovascular mortality in T2D-CLI patients. An altered paracrine signaling conveys antiangiogenic and proapoptotic features from CD34+ cells to the endothelium. This damaging interaction may increase the risk for life-threatening complications.

Bone marrow pericyte dysfunction in individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Previous studies have shown that diabetes mellitus destabilises the integrity of the microvasculature in different organs by damaging the interaction between pericytes and endothelial cells. In bone marrow, pericytes exert trophic functions on endothelial cells and haematopoietic cells through paracrine mechanisms. However, whether bone marrow pericytes are a target of diabetes-induced damage remains unknown. Here, we investigated whether type 2 diabetes can affect the abundance and function of bone marrow pericytes. METHODS: We conducted an observational clinical study comparing the abundance and molecular/functional characteristics of CD146+ pericytes isolated from the bone marrow of 25 individuals without diabetes and 14 individuals with uncomplicated type 2 diabetes, referring to our Musculoskeletal Research Unit for hip reconstructive surgery. RESULTS: Immunohistochemistry revealed that diabetes causes capillary rarefaction and compression of arteriole size in bone marrow, without changing CD146+ pericyte counts. These data were confirmed by flow cytometry on freshly isolated bone marrow cells. We then performed an extensive functional and molecular characterisation of immunosorted CD146+ pericytes. Type 2 diabetes caused a reduction in pericyte proliferation, viability, migration and capacity to support in vitro angiogenesis, while inducing apoptosis. AKT is a key regulator of the above functions and its phosphorylation state is reportedly reduced in the bone marrow endothelium of individuals with diabetes. Surprisingly, we could not find a difference in AKT phosphorylation (at either Ser473 or Thr308) in bone marrow pericytes from individuals with and without diabetes. Nonetheless, the angiocrine signalling reportedly associated with AKT was found to be significantly downregulated, with lower levels of fibroblast growth factor-2 (FGF2) and C-X-C motif chemokine ligand 12 (CXCL12), and activation of the angiogenesis inhibitor angiopoietin 2 (ANGPT2). Transfection with the adenoviral vector carrying the coding sequence for constitutively active myristoylated AKT rescued functional defects and angiocrine signalling in bone marrow pericytes from diabetic individuals. Furthermore, an ANGPT2 blocking antibody restored the capacity of pericytes to promote endothelial networking. CONCLUSIONS/INTERPRETATION: This is the first demonstration of pericyte dysfunction in bone marrow of people with type 2 diabetes. An altered angiocrine signalling from pericytes may participate in bone marrow microvascular remodelling in individuals with diabetes.

Concise Review: The Regenerative Journey of Pericytes Toward Clinical Translation.

Coronary artery disease (CAD) is the single leading cause of death worldwide. Advances in treatment and management have significantly improved patient outcomes. On the other hand, although mortality rates have decreased, more people are left with sequelae that require additional treatment and hospitalization. Moreover, patients with severe nonrevascularizable CAD remain with only the option of heart transplantation, which is limited by the shortage of suitable donors. In recent years, cell-based regenerative therapy has emerged as a possible alternative treatment, with several regenerative medicinal products already in the clinical phase of development and others emerging as competitive preclinical solutions. Recent evidence indicates that pericytes, the mural cells of blood microvessels, represent a promising therapeutic candidate. Pericytes are abundant in the human body, play an active role in angiogenesis, vessel stabilization and blood flow regulation, and possess the capacity to differentiate into multiple cells of the mesenchymal lineage. Moreover, early studies suggest a robustness to hypoxic insult, making them uniquely equipped to withstand the ischemic microenvironment. This review summarizes the rationale behind pericyte-based cell therapy and the progress that has been made toward its clinical application. We present the different sources of pericytes and the case for harvesting them from tissue leftovers of cardiovascular surgery. We also discuss the healing potential of pericytes in preclinical animal models of myocardial ischemia (MI) and current practices to upgrade the production protocol for translation to the clinic. Standardization of these procedures is of utmost importance, as lack of uniformity in cell manufacturing may influence clinical outcome. Stem Cells 2018;36:1295-1310.

MCP-1 Feedback Loop Between Adipocytes and Mesenchymal Stromal Cells Causes Fat Accumulation and Contributes to Hematopoietic Stem Cell Rarefaction in the Bone Marrow of Patients With Diabetes.

Fat accumulates in bone marrow (BM) of patients with diabetes. In this study, we investigated the mechanisms and consequences of this phenomenon. BM mesenchymal stromal cells (BM-MSCs) from patients with type 2 diabetes (T2D) constitutively express adipogenic markers and robustly differentiate into adipocytes (ADs) upon in vitro induction as compared with BM-MSCs from subjects without diabetes. Moreover, BM-ADs from subjects with T2D (T2D BM-ADs) paracrinally stimulate a transcriptional adipogenic program in BM-MSCs. Antagonism of MCP-1, a chemokine pivotally expressed in T2D BM-ADs, prevented the T2D BM-AD secretome from converting BM-MSCs into ADs. Mechanistic validation of human data was next performed in an obese T2D mouse model. Systemic antagonism of MCP-1 improved metabolic control, reduced BM fat, and increased osteocyte density. It also indirectly re-established the abundance of long-term versus short-term hematopoietic stem cells. We reveal a diabetic feedback loop in which 1) BM-MSCs are constitutively inclined to make ADs, and 2) mature BM-ADs, via secreted MCP-1, relentlessly fuel BM-MSC determination into new fat. Pharmacological inhibition of MCP-1 signaling can contrast this vicious cycle, restoring, at least in part, the balance between adipogenesis and hematopoiesis in BM from subjects with T2D.

Activation of the Pro-Oxidant PKCßII-p66Shc Signaling Pathway Contributes to Pericyte Dysfunction in Skeletal Muscles of Patients With Diabetes With Critical Limb Ischemia.

Critical limb ischemia (CLI), foot ulcers, former amputation, and impaired regeneration are independent risk factors for limb amputation in subjects with diabetes. The present work investigates whether and by which mechanism diabetes negatively impacts on functional properties of muscular pericytes (MPs), which are resident stem cells committed to reparative angiomyogenesis. We obtained muscle biopsy samples from patients with diabetes who were undergoing major limb amputation and control subjects. Diabetic muscles collected at the rim of normal tissue surrounding the plane of dissection showed myofiber degeneration, fat deposition, and reduction of MP vascular coverage. Diabetic MPs (D-MPs) display ultrastructural alterations, a differentiation bias toward adipogenesis at the detriment of myogenesis and an inhibitory activity on angiogenesis. Furthermore, they have an imbalanced redox state, with downregulation of the antioxidant enzymes superoxide dismutase 1 and catalase, and activation of the pro-oxidant protein kinase C isoform ß-II (PKCßII)-dependent p66Shc signaling pathway. A reactive oxygen species scavenger or, even more effectively, clinically approved PKCßII inhibitors restore D-MP angiomyogenic activity. Inhibition of the PKCßII-dependent p66Shc signaling pathway could represent a novel therapeutic approach for the promotion of muscle repair in individuals with diabetes.

Sensory neuropathy hampers nociception-mediated bone marrow stem cell release in mice and patients with diabetes.

AIMS/HYPOTHESIS: Upon tissue injury, peripheral sensory neurons release nociceptive factors (e.g. substance P [SP]), which exert local and systemic actions including the recruitment of bone marrow (BM)-derived haematopoietic stem and progenitor cells (HSPCs) endowed with paracrine pro-angiogenic properties. We herein explore whether diabetic neuropathy interferes with these phenomena. METHODS: We first investigated the presence of sensory neuropathy in the BM of patients with type 2 diabetes by immunohistochemistry and morphometry analyses of nerve size and density and assessment of SP release by ELISA. We next analysed the association of sensory neuropathy with altered HSPC release under ischaemia or following direct stimulation with granulocyte colony-stimulating factor (G-CSF). BM and circulating HSPCs expressing the neurokinin 1 receptor (NK1R), which is the main SP receptor, were measured by flow cytometry. We finally assessed whether an altered modulation of SP secretion interferes with the mobilisation and homing of NK1R-HSPCs in a mouse model of type 2 diabetes after limb ischaemia (LI). RESULTS: Nociceptive fibres were reduced in the BM of patients and mice with type 2 diabetes. Patients with neuropathy showed a remarkable reduction in NK1R-HSPC mobilisation under ischaemia or upon G-CSF stimulation. Following LI, diabetic mice manifested an altered SP gradient between BM, peripheral blood and limb muscles, accompanied by a depressed recruitment of NK1R-HSPCs to the ischaemic site. CONCLUSIONS/INTERPRETATION: Sensory neuropathy translates into defective liberation and homing of reparative HSPCs. Nociceptors may represent a new target for treatment of diabetic complications.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells.

Characterization of lymphatic endothelial cells from the respiratory system may be crucial to investigate the role of the lymphatic system in the normal and diseased lung. We describe a simple and inexpensive method to harvest, isolate, and expand lymphatic endothelial cells from the human lung (HL-LECs). Fifty-five samples of healthy lung selected from patients undergoing lobectomy were studied. A two-step purification tool, based on paramagnetic sorting with monoclonal antibodies to CD31 and Podoplanin, was employed to select a pure population of HL-LECs. The purity of HL-LECs was assessed by morphologic criteria, immunocytochemistry, flow cytometry, and functional assays. Interestingly, these cells retain in vitro several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation. HL-LECs represent a clinically relevant cellular substrate to study lymphatic biology, lymphoangiogenesis, interaction with microbial agents, wound healing, and anticancer therapy.