HCG18, LEF1AS1 and lncCEACAM21 as biomarkers of disease severity in the peripheral blood mononuclear cells of COVID-19 patients.

BACKGROUND: Even after 3 years from SARS-CoV-2 identification, COVID-19 is still a persistent and dangerous global infectious disease. Significant improvements in our understanding of the disease pathophysiology have now been achieved. Nonetheless, reliable and accurate biomarkers for the early stratification of COVID-19 severity are still lacking. Long noncoding RNAs (LncRNAs) are ncRNAs longer than 200 nucleotides, regulating the transcription and translation of protein-coding genes and they can be found in the peripheral blood, thus holding a promising biomarker potential. Specifically, peripheral blood mononuclear cells (PBMCs) have emerged as a source of indirect biomarkers mirroring the conditions of tissues: they include monocytes, B and T lymphocytes, and natural killer T cells (NKT), being highly informative for immune-related events. METHODS: We profiled by RNA-Sequencing a panel of 2906 lncRNAs to investigate their modulation in PBMCs of a pilot group of COVID-19 patients, followed by qPCR validation in 111 hospitalized COVID-19 patients. RESULTS: The levels of four lncRNAs were found to be decreased in association with COVID-19 mortality and disease severity: HLA Complex Group 18-242 and -244 (HCG18-242 and HCG18-244), Lymphoid Enhancer Binding Factor 1-antisense 1 (LEF1-AS1) and lncCEACAM21 (i.e. ENST00000601116.5, a lncRNA in the CEACAM21 locus). Interestingly, these deregulations were confirmed in an independent patient group of hospitalized patients and by the re-analysis of publicly available single-cell transcriptome datasets. The identified lncRNAs were expressed in all of the PBMC cell types and inversely correlated with the neutrophil/lymphocyte ratio (NLR), an inflammatory marker. In vitro, the expression of LEF1-AS1 and lncCEACAM21 was decreased upon THP-1 monocytes exposure to a relevant stimulus, hypoxia. CONCLUSION: The identified COVID-19-lncRNAs are proposed as potential innovative biomarkers of COVID-19 severity and mortality.

Cardiovascular complications of diabetes: role of non-coding RNAs in the crosstalk between immune and cardiovascular systems.

Diabetes mellitus, a group of metabolic disorders characterized by high levels of blood glucose caused by insulin defect or impairment, is a major risk factor for cardiovascular diseases and related mortality. Patients with diabetes experience a state of chronic or intermittent hyperglycemia resulting in damage to the vasculature, leading to micro- and macro-vascular diseases. These conditions are associated with low-grade chronic inflammation and accelerated atherosclerosis. Several classes of leukocytes have been implicated in diabetic cardiovascular impairment. Although the molecular pathways through which diabetes elicits an inflammatory response have attracted significant attention, how they contribute to altering cardiovascular homeostasis is still incompletely understood. In this respect, non-coding RNAs (ncRNAs) are a still largely under-investigated class of transcripts that may play a fundamental role. This review article gathers the current knowledge on the function of ncRNAs in the crosstalk between immune and cardiovascular cells in the context of diabetic complications, highlighting the influence of biological sex in such mechanisms and exploring the potential role of ncRNAs as biomarkers and targets for treatments. The discussion closes by offering an overview of the ncRNAs involved in the increased cardiovascular risk suffered by patients with diabetes facing Sars-CoV-2 infection.

Results of a prospective observational study of autologous peripheral blood mononuclear cell therapy for no-option critical limb-threatening ischemia and severe diabetic foot ulcers.

BACKGROUND: Cell therapy with autologous peripheral blood mononuclear cells (PB-MNCs) may help restore limb perfusion in patients with diabetes mellitus and critical limb-threatening ischemia (CLTI) deemed not eligible for revascularization procedures and consequently at risk for major amputation (no-option). Fundamental is to establish its clinical value and to identify candidates with a greater benefit over time. Assessing the frequency of PB circulating angiogenic cells and extracellular vesicles (EVs) may help in guiding candidate selection. METHODS: We conducted a prospective, non-controlled, observational study on no-option CLTI diabetic patients that underwent intramuscular PB-MNCs therapy, which consisted of more cell treatments repeated a maximum of three times. The primary endpoint was amputation rate at 1 year following the first treatment with PB-MNCs. We evaluated ulcer healing, walking capability, and mortality during the follow-up period. We assessed angiogenic cells and EVs at baseline and after each cell treatment, according to primary outcome and tissue perfusion at the last treatment [measured as transcutaneous oxygen pressure (TcPO2)]. RESULTS: 50 patients were consecutively enrolled and the primary endpoint was 16%. TcPO2 increased after PB-MNCs therapy (17.2 ± 11.6 vs 39.1 ± 21.8 mmHg, p < .0001), and ulcers healed with back-to-walk were observed in 60% of the study population (88% of survivors) during follow-up (median 1.5 years). Patients with a high level of TcPO2 (≥ 40 mmHg) after the last treatment showed a high frequency of small EVs at enrollment. CONCLUSIONS: In no-option CLTI diabetic patients, PB-MNCs therapy led to an improvement in tissue perfusion, a high rate of healing, and back-to-walk. Coupling circulating cellular markers of angiogenesis could help in the identification of patients with a better clinical benefit over time.

Impaired Regeneration Contributes to Poor Outcomes in Diabetic Peripheral Artery Disease.

Diabetes mellitus increases the risk and accelerates the course of peripheral artery disease, making patients more susceptible to ischemic events and infections and delaying tissue healing. Current understanding of pathogenic mechanisms is mainly based on the negative influence of diabetes mellitus on atherosclerotic disease and inflammation. In recent years, the novel concept that diabetes mellitus can impinge on endogenous regenerative processes has been introduced. Diabetes mellitus affects regeneration at the local level, disturbing proper angiogenesis, collateral artery formation, and muscle repair. Recent evidence indicates that an impairment in vascular mural cells, alias pericytes, may participate in diabetic peripheral vasculopathy. Moreover, the bone marrow undergoes a global remodeling, consisting of microvessels and sensory neurons rarefaction and fat accumulation, which creates a hostile microenvironment for resident stem cells. Bone marrow remodeling is also responsible for detrimental systemic effects. In particular, the aid of reparative cells from the bone marrow is compromised: these elements are released in an improper manner and become harmful vectors of inflammatory and antiangiogenic molecules and noncoding RNAs. This new understanding of impaired regeneration is inspiring new therapeutic options for the treatment of ischemic complications in people with diabetes mellitus.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

Bone marrow as a target and accomplice of vascular complications in diabetes.

Peripheral vascular complications are common in diabetic patients. While pathogenic mechanisms have received much consideration, only recently regenerative processes captured attention. There is now a consensus that the bone marrow is a source of reparative cells and that this healing mechanism is lost in people with diabetes, especially in those suffering from ischemic complications. This failure was thought to occur due to a negative impact of diabetes on the mobilization of stem/progenitor cells with angiogenic properties from the bone marrow to the circulation. Moreover, those patients showing severely reduced bone marrow cell mobilization also bared a very high risk for adverse cardiovascular events. More recently, the structural integrity of the bone marrow was recognized to be altered because of the rarefaction of local microvasculature and innervation, thus mirroring anatomical features that typically occur in peripheral tissues. Ensuing hypoxia, nutrient starvation, and creation of an acidic and oxidative environment concur in causing the depletion of stem/progenitor cells in the endosteal niche and in forcing stromal cells to activate an adipogenesis program. Moreover, stem/progenitor cells acquire a pathogenic phenotype and, once mobilized, can pass harmful signalling molecules to vascular cells of peripheral tissues thereby contributing to ischemic complications. These new pieces of evidence indicate that the bone marrow should deserve more attention in the current care of critical limb ischemia and diabetic foot. Owing to powerful reserve capacities, the bone marrow integrity could be preserved and even rescued using rehabilitation programs and pharmacological treatments with consequent benefit for local and whole-organism homeostasis.

Bone marrow fat: friend or foe in people with diabetes mellitus?

Global trends in the prevalence of overweight and obesity put the adipocyte in the focus of huge medical interest. This review highlights a new topic in adipose tissue biology, namely the emerging pathogenic role of fat accumulation in bone marrow (BM). Specifically, we summarize current knowledge about the origin and function of BM adipose tissue (BMAT), provide evidence for the association of excess BMAT with diabetes and related cardiovascular complications, and discuss potential therapeutic approaches to correct BMAT dysfunction. There is still a significant uncertainty about the origins and function of BMAT, although several subpopulations of stromal cells have been suggested to have an adipogenic propensity. BM adipocytes are higly plastic and have a distinctive capacity to secrete adipokines that exert local and endocrine functions. BM adiposity is abundant in elderly people and has therefore been interpreted as a component of the whole-body ageing process. BM senescence and BMAT accumulation has been also reported in patients and animal models with Type 2 diabetes, being more pronounced in those with ischaemic complications. Understanding the mechanisms responsible for excess and altered function of BMAT could lead to new treatments able to preserve whole-body homeostasis.

Role of TPBG (Trophoblast Glycoprotein) Antigen in Human Pericyte Migratory and Angiogenic Activity.

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.

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.

Correction to: Bone marrow pericyte dysfunction in individuals with type 2 diabetes.

Unfortunately, three errors were made in the conversion of HbA1c to per cent values.

Circulating microRNA-21 is an early predictor of ROS-mediated damage in subjects with high risk of developing diabetes and in drug-naïve T2D.

BACKGROUND: Impaired glucose tolerance (IGT) is a risk factor for the development of diabetes and related complications that ensue. Early identification of at-risk individuals might be beneficial to reduce or delay the progression of diabetes and its related complications. Recently, microRNAs emerged as potential biomarkers of diseases. The aim of the present study was to evaluate microRNA-21 as a potential biomarker for the risk of developing diabetes in adults with IGT and to investigate its downstream effects as the generation of reactive oxygen species (ROS), the induction of manganese-superoxide dismutase-2 (SOD2), and the circulating levels of 4-HNE (4-hydroxynonenal). METHODS: To evaluate the prognostic and predictive values of plasmatic microRNA-21 in identifying metabolic derangements, we tested a selected cohort (n = 115) of subjects enrolled in the DIAPASON Study, whom were selected on ADA criteria for 2hPG. Statistical analysis was performed using ANOVA or the Kruskal-Wallis test as appropriate. ROC curves were drawn for diagnostic accuracy of the tests; positive and negative predictive values were performed, and Youden's index was used to seek the cut-off optimum truncation point. ROS, SOD2 and 4-HNE were also evaluated. RESULTS: We observed significant upregulation of microRNA-21 in IGT and in T2D subjects, and microRNA-21 was positively correlated with glycaemic parameters. Diagnostic performance of microRNA-21 was high and accurate. We detected significant overproduction of ROS by electron paramagnetic resonance (EPR), significant accumulation of the lipid peroxidation marker 4-HNE, and defective SOD2 antioxidant response in IGT and newly diagnosed, drug-naïve T2D subjects. In addition, ROC curves demonstrated the diagnostic accuracy of markers used. CONCLUSIONS: our data demonstrate that microRNA-21 is associated with prediabetic status and exhibits predictive value for early detection of glucose imbalances. These data could provide novel clues for miR-based biomarkers to evaluate diabetes.

MicroRNA-532-5p Regulates Pericyte Function by Targeting the Transcription Regulator BACH1 and Angiopoietin-1.

MicroRNAs regulate endothelial function and angiogenesis, but their implication in pericyte biology remains undetermined. A PCR array, covering a panel of 379 human microRNAs, showed microRNA-532-5p to be one of the most differentially modulated by hypoxia, which was confirmed by qPCR in both skeletal muscle and adventitial pericytes. Furthermore, microRNA-532-5p was upregulated in murine muscular pericytes early after experimentally induced ischemia, decreasing below baseline after reperfusion. Transfection of human pericytes with anti-microRNA, microRNA-mimic, or controls indicates microRNA-532-5p modulates pro-angiogenic activity via transcriptional regulation of angiopoietin-1. Tie-2 blockade abrogated the ability of microRNA-532-5p-overexpressing pericytes to promote endothelial network formation in vitro. However, angiopoietin-1 is not a direct target of microRNA-532-5p. In silico analysis of microRNA-532-5p inhibitory targets associated with angiopoietin-1 transcription indicated three potential candidates, BACH1, HIF1AN, and EGLN1. Binding of microRNA-532-5p to the BACH1 3' UTR was confirmed by luciferase assay. MicroRNA-532-5p silencing increased BACH1, while a microRNA-532-5p mimic decreased expression. Silencing of BACH1 modulated angiopoietin-1 gene and protein expression. ChIP confirmed BACH1 transcriptional regulation of angiopoietin-1 promoter. Finally, microRNA-532-5p overexpression increased pericyte coverage in an in vivo Matrigel assay, suggesting its role in vascular maturation. This study provides a new mechanistic understanding of the transcriptional program orchestrating angiopoietin-1/Tie-2 signaling in human pericytes.

miR-210 Enhances the Therapeutic Potential of Bone-Marrow-Derived Circulating Proangiogenic Cells in the Setting of Limb Ischemia.

Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia.

Human Pericardial Fluid Contains Exosomes Enriched with Cardiovascular-Expressed MicroRNAs and Promotes Therapeutic Angiogenesis.

The pericardial fluid (PF) is contained in the pericardial sac surrounding the heart. MicroRNA (miRNA) exchange via exosomes (endogenous nanoparticles) contributes to cell-to-cell communication. We investigated the hypotheses that the PF is enriched with miRNAs secreted by the heart and that it mediates vascular responses through exosome exchange of miRNAs. The study was developed using leftover material from aortic valve surgery. We found that in comparison with peripheral plasma, the PF contains exosomes enriched with miRNAs co-expressed in patients' myocardium and vasculature. At a functional level, PF exosomes improved survival, proliferation, and networking of cultured endothelial cells (ECs) and restored the angiogenic capacity of ECs depleted (via Dicer silencing) of their endogenous miRNA content. Moreover, PF exosomes improved post-ischemic blood flow recovery and angiogenesis in mice. Mechanistically, (1) let-7b-5p is proangiogenic and inhibits its target gene, TGFBR1, in ECs; (2) PF exosomes transfer a functional let-7b-5p to ECs, thus reducing their TGFBR1 expression; and (3) let-7b-5p depletion in PF exosomes impairs the angiogenic response to these nanoparticles. Collectively, our data support the concept that PF exosomes orchestrate vascular repair via miRNA transfer.

Genetic Analysis Reveals a Longevity-Associated Protein Modulating Endothelial Function and Angiogenesis.

RATIONALE: Long living individuals show delay of aging, which is characterized by the progressive loss of cardiovascular homeostasis, along with reduced endothelial nitric oxide synthase activity, endothelial dysfunction, and impairment of tissue repair after ischemic injury. OBJECTIVE: Exploit genetic analysis of long living individuals to reveal master molecular regulators of physiological aging and new targets for treatment of cardiovascular disease. METHODS AND RESULTS: We show that the polymorphic variant rs2070325 (Ile229Val) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) associates with exceptional longevity, under a recessive genetic model, in 3 independent populations. Moreover, the expression of BPIFB4 is instrumental to maintenance of cellular and vascular homeostasis through regulation of protein synthesis. BPIFB4 phosphorylation/activation by protein-kinase-R-like endoplasmic reticulum kinase induces its complexing with 14-3-3 and heat shock protein 90, which is facilitated by the longevity-associated variant. In isolated vessels, BPIFB4 is upregulated by mechanical stress, and its knock-down inhibits endothelium-dependent vasorelaxation. In hypertensive rats and old mice, gene transfer of longevity-associated variant-BPIFB4 restores endothelial nitric oxide synthase signaling, rescues endothelial dysfunction, and reduces blood pressure levels. Furthermore, BPIFB4 is implicated in vascular repair. BPIFB4 is abundantly expressed in circulating CD34(+) cells of long living individuals, and its knock-down in endothelial progenitor cells precludes their capacity to migrate toward the chemoattractant SDF-1. In a murine model of peripheral ischemia, systemic gene therapy with longevity-associated variant-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization, and reperfusion of the ischemic muscle. CONCLUSIONS: Longevity-associated variant-BPIFB4 may represent a novel therapeutic tool to fight endothelial dysfunction and promote vascular reparative processes.

The genetics of exceptional longevity identifies new druggable targets for vascular protection and repair.

Therapeutic angiogenesis is a relatively new medical strategy in the field of cardiovascular diseases. The underpinning concept is that angiogenic growth factors or proangiogenic cells could be exploited therapeutically in cardiovascular patients to enhance native revascularization responses to an ischemic insult, thereby accelerating tissue healing. The initial enthusiasm generated by preclinical studies has been tempered by the modest success of clinical trials assessing therapeutic angiogenesis. Similarly, proangiogenic cell therapy has so far not maintained the original promises. Intriguingly, the current trend is to consider regeneration as a prerogative of the youngest organism. Consequentially, the embryonic and foetal models are attracting much attention for clinical translation into corrective modalities in the adulthood. Scientists seem to undervalue the lesson from Mother Nature, e.g. all humans are born young but very few achieve the goal of an exceptional healthy longevity. Either natural experimentation is driven by a supreme intelligence or stochastic phenomena, one has to accept the evidence that healthy longevity is the fruit of an evolutionary process lasting million years. It is therefore extremely likely that results of this natural experimentation are more reliable and translatable than the intensive, but very short human investigation on mechanisms governing repair and regeneration. With this preamble in mind, here we propose to shift the focus from the very beginning to the very end of human life and thus capture the secret of prolonged health span to improve well-being in the adulthood.

Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.

OBJECTIVE: We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. APPROACH AND RESULTS: Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)-derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×10(5) cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. CONCLUSIONS: DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

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.

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.

BACKGROUND: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. RESULTS: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ1-8Met-MTGase from NP668/1 and long constitutive LacZ1-8PNP1-20Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ1-8PNP1-20Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. CONCLUSIONS: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ1-8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.

MicroRNA-15a and microRNA-16 impair human circulating proangiogenic cell functions and are increased in the proangiogenic cells and serum of patients with critical limb ischemia.

RATIONALE: Circulating proangiogenic cells (PACs) support postischemic neovascularization. Cardiovascular disease and diabetes mellitus impair PAC regenerative capacities via molecular mechanisms that are not fully known. We hypothesize a role for microRNAs (miRs). Circulating miRs are currently investigated as potential diagnostic and prognostic biomarkers. OBJECTIVE: The objectives were the following: (1) to profile miR expression in PACs from critical limb ischemia (CLI) patients; (2) to demonstrate that miR-15a and miR-16 regulate PAC functions; and (3) to characterize circulating miR-15a and miR-16 and to investigate their potential biomarker value. METHODS AND RESULTS: Twenty-eight miRs potentially able to modulate angiogenesis were measured in PACs from CLI patients with and without diabetes mellitus and controls. miR-15a and miR-16 were further analyzed. CLI-PACs expressed higher level of mature miR-15a and miR-16 and of the primary transcript pri-miR-15a/16-1. miR-15a/16 overexpression impaired healthy PAC survival and migration. Conversely, miR-15a/16 inhibition improved CLI-PAC-defective migration. Vascular endothelial growth factor-A and AKT-3 were validated as direct targets of the 2 miRs, and their protein levels were reduced in miR-15a/16-overexpressing healthy PACs and in CLI-PACs. Transplantation of healthy PACs ex vivo-engineered with anti-miR-15a/16 improved postischemic blood flow recovery and muscular arteriole density in immunodeficient mice. miR-15a and miR-16 were present in human blood, including conjugated to argonaute-2 and in exosomes. Both miRs were increased in the serum of CLI patients and positively correlated with amputation after restenosis at 12 months postrevascularization of CLI type 2 diabetes mellitus patients. Serum miR-15a additionally correlated with restenosis at follow-up. CONCLUSIONS: Ex vivo miR-15a/16 inhibition enhances PAC therapeutic potential, and circulating miR-15a and miR-16 deserves further investigation as a prognostic biomarker in CLI patients undergoing revascularization.