Metataxonomic analysis of milk microbiota in the bovine subclinical mastitis.

Subclinical mastitis is one of the most widespread diseases affecting dairy herds with detrimental effects on animal health, milk productivity, and quality. Despite its multifactorial nature, the presence of pathogenic bacteria is regarded one of the main drivers of subclinical mastitis, causing a disruption of the homeostasis of the bovine milk microbial community. However, bovine milk microbiota alterations associated with subclinical mastitis still represents a largely unexplored research area. Therefore, the species-level milk microbiota of a total of 75 milk samples, collected from both healthy and subclinical mastitis-affected cows from two different stables, was deeply profiled through an ITS, rather than a traditional, and less informative, 16S rRNA gene microbial profiling. Surprisingly, the present pilot study not only revealed that subclinical mastitis is characterized by a reduced biodiversity of the bovine milk microbiota, but also that this disease does not induce standard alterations of the milk microbial community across stables. In addition, a flow cytometry-based total bacterial cell enumeration highlighted that subclinical mastitis is accompanied by a significant increment in the number of milk microbial cells. Furthermore, the combination of the metagenomic and flow cytometry approaches allowed to identify different potential microbial marker strictly correlated with subclinical mastitis across stables.

The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy.

AIMS: The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene-environment interaction. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector improves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV-BPIFB4 gene could address the unmet therapeutic need to delay the heart's spontaneous ageing. METHODS AND RESULTS: Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supplementation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. CONCLUSIONS: We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart's ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people.

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.

Hematopoietic progenitor cell liabilities and alarmins S100A8/A9-related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults.

Frailty affects the physical, cognitive, and social domains exposing older adults to an increased risk of cardiovascular disease and death. The mechanisms linking frailty and cardiovascular outcomes are mostly unknown. Here, we studied the association of abundance (flow cytometry) and gene expression profile (RNAseq) of stem/progenitor cells (HSPCs) and molecular markers of inflammaging (ELISA) with the cardiorespiratory phenotype and prospective adverse events of individuals classified according to levels of frailty. Two cohorts of older adults were enrolled in the study. In a cohort of pre-frail 35 individuals (average age: 75 years), a physical frailty score above the median identified subjects with initial alterations in cardiorespiratory function. RNA sequencing revealed S100A8/A9 upregulation in HSPCs from the bone marrow (>10-fold) and peripheral blood (>200-fold) of individuals with greater physical frailty. Moreover higher frailty was associated with increased alarmins S100A8/A9 and inflammatory cytokines in peripheral blood. We then studied a cohort of 104 more frail individuals (average age: 81 years) with multidomain health deficits. Reduced levels of circulating HSPCs and increased S100A8/A9 concentrations were independently associated with the frailty index. Remarkably, low HSPCs and high S100A8/A9 simultaneously predicted major adverse cardiovascular events at 1-year follow-up after adjustment for age and frailty index. In conclusion, inflammaging characterized by alarmin and pro-inflammatory cytokines in pre-frail individuals is mirrored by the pauperization of HSPCs in frail older people with comorbidities. S100A8/A9 is upregulated within HSPCs, identifying a phenotype that associates with poor cardiovascular outcomes.

Modulation of soluble receptor for advanced glycation end products isoforms and advanced glycation end products in long-living individuals.

Background: Circulating levels of soluble receptor for advanced glycation end products (sRAGE) and advanced glycation end products (AGEs) correlate with aging/cardiovascular risk, which is delayed in long-living individuals (LLIs). AGEs/sRAGE isoforms (cleaved RAGE [cRAGE] and secretory RAGE [esRAGE]) ratio is a valuable marker for disease risk. Results: We evaluated circulating sRAGE isoforms, and AGEs in LLIs (n = 95; 90-105 years) and controls (n = 94; 11-89 years). cRAGE decreased with age in controls and further declined in LLIs. esRAGE increased in LLIs. AGEs rose with age in controls and decreased in LLIs that were characterized by a lower AGEs/sRAGE ratio. Notably, cRAGE and AGE/esRAGE ratio better discriminated controls from LLIs. Conclusion: circulating cRAGE could be considered a reliable marker of chronological age while esRAGE a protective factor for longevity.

Lay abstract Aging is the major risk factor for disease development. Long-living individuals (LLIs) are subjects older than 90 years that represent an invaluable model to study mechanisms underpinning longevity and healthy aging. Circulating levels of soluble receptor for advanced glycation end products (sRAGE) change with aging and can forecast the cardiovascular risk, which is reduced in centenarians. sRAGE is composed of two isoforms, the cleaved RAGE (cRAGE) and the secretory RAGE (esRAGE), that are known to inhibit the oxidative stress and inflammatory activities of their ligands such the advanced glycation end products (AGEs). In this study, we measured the plasmatic levels of both sRAGE isoforms and AGEs in LLIs (90­105 years) and control subjects (11­89 years). We found that cRAGE decreases with age in controls and LLIs. esRAGE increases in LLIs and AGEs increase in controls with age but decrease in LLIs. AGEs/esRAGE ratio and cRAGE were able to discriminate controls from LLIs. Hence, LLIs are characterized by a lower AGEs/sRAGE ratio, due to esRAGE increase and AGEs reduction that may explain their reduced cardiovascular and metabolic risk. Besides, circulating cRAGE could be considered a reliable marker of chronological age, while esRAGE a protective factor associated with longevity.

Selective photoredox direct arylations of aryl bromides in water in a microfluidic reactor.

Carrying out photoredox direct arylation couplings between aryl halides and aryls in aqueous solutions of surfactants enables unprecedented selectivity with respect to the competing dehalogenation process, thanks to the partition coefficient of the selected sacrificial base. The use of a microfluidic reactor dramatically improves the reaction time, without eroding the yields and selectivity. The design of a metal free sensitizer, which also acts as the surfactant, sizeably improves the overall sustainability of arylation reactions and obviates the need for troublesome purification from traces of metal catalysts. The generality of the method is investigated over a range of halides carrying a selection of electron withdrawing and electron donating substituents.

Microfluidic Synthesis of Hybrid TiO2-Anisotropic Gold Nanoparticles with Visible and Near-Infrared Activity.

Anisotropic gold nanoparticles (AuNPs), with their unique physical and optical properties, are emerging as smart and key nanomaterials and are being exploited in many crucial fields. To further improve their range of action, anisotropic AuNPs have been coupled with semiconductors, mainly TiO2 (titania), receiving great interest as powerful platforms both in biomedicine and in catalytic applications. Such hybrid nanoparticles show new properties that arise from the synergic action of the components and rely on NP size, morphology, and arrangement. Therefore, continuous advances in design and fabrication of new hybrid titania@gold NPs (TiO2@AuNPs) are urgent and highly desirable. Here, we propose an effective protocol to produce multibranched AuNPs covered by a controlled TiO2 thin layer, exploiting a one-pot microfluidic process. The proposed method allows the in-flow and reliable synthesis of titania-functionalized-anisotropic gold nanoparticles by avoiding the use of toxic surfactants and controlling the titania shell formation. TiO2@AuNPs have been fully characterized in terms of morphology, stability, and biocompatibility, and their activity in photocatalysis has been tested and verified.

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.

Circulating MicroRNA-15a Associates With Retinal Damage in Patients With Early Stage Type 2 Diabetes.

Circulating microRNAs are potential biomarkers of type 2 diabetes mellitus (T2DM) and related complications. Here, we investigated the association of microRNA-15a with early retinal damage in T2DM. A cohort of untreated subjects screened for intermediate/high risk of T2DM, according to a score assessment questionnaire, and then recognized to have a normal (NGT) or impaired (IGT) glucose tolerance or T2DM was studied. The thickness of the ganglion cell complex (GCC), an early marker of retinal degeneration anteceding overt retinopathy was assessed by Optical Coherence Tomography. Total and extracellular vesicles (EV)-associated microRNA-15a quantity was measured in plasma by real time PCR. MicroRNA-15a level was significantly higher in subjects with IGT and T2DM compared with NGT. MicroRNA-15a abundance was correlated to body mass index and classical diabetes biomarkers, including fasting glucose, HbA1c, insulinemia, and HOMA-IR. Moreover, GCC thickness was significantly reduced in IGT and T2DM subjects compared with NGT controls. Importantly, total microRNA-15a correlated with GCC in IGT subjects, while in T2DM subjects, EV-microRNA-15a negatively correlated with GCC, suggesting that microRNA-15a may monitor initial retinal damage. The assessment of plasma microRNA-15a may help refining risk assessment and secondary prevention in patients with preclinical T2DM.

Corrigendum: 3D hydrogel environment rejuvenates aged pericytes for skeletal muscle tissue engineering.

[This corrects the article DOI: 10.3389/fphys.2014.00203.].

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.

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.

Central role of the p53 pathway in the noncoding-RNA response to oxidative stress.

Oxidative stress plays a fundamental role in many conditions. Specifically, redox imbalance inhibits endothelial cell (EC) growth, inducing cell death and senescence. We used global transcriptome profiling to investigate the involvement of noncoding-RNAs in these phenotypes. By RNA-sequencing, transcriptome changes were analyzed in human ECs exposed to H2O2, highlighting a pivotal role of p53-signaling. Bioinformatic analysis and validation in p53-silenced ECs, identified several p53-targets among both mRNAs and long noncoding-RNAs (lncRNAs), including MALAT1 and NEAT1. Among microRNAs (miRNAs), miR-192-5p was the most induced by H2O2 treatment, in a p53-dependent manner. Down-modulated mRNA-targets of miR-192-5p were involved in cell cycle, DNA repair and stress response. Accordingly, miR-192-5p overexpression significantly decreased EC proliferation, inducing cell death. A central role of the p53-pathway was also confirmed by the analysis of differential exon usage: Upon H2O2 treatment, the expression of p53-dependent 5'-isoforms of MDM2 and PVT1 increased selectively. The transcriptomic alterations identified in H2O2-treated ECs were also observed in other physiological and pathological conditions where redox control plays a fundamental role, such as ECs undergoing replicative senescence, skeletal muscles of critical limb-ischemia patients and the peripheral-blood mononuclear cells of long-living individuals. Collectively, these findings indicate a prominent role of noncoding-RNAs in oxidative stress response.

A unique plasma microRNA profile defines type 2 diabetes progression.

A major unmet medical need to better manage Type 2 Diabetes (T2D) is the accurate disease prediction in subjects who show glucose dysmetabolism, but are not yet diagnosed as diabetic. We investigated the possibility to predict/monitor the progression to T2D in these subjects by retrospectively quantifying blood circulating microRNAs in plasma of subjects with i) normal glucose tolerance (NGT, n = 9); ii) impaired glucose tolerance (IGT, n = 9), divided into non-progressors (NP, n = 5) and progressors (P, n = 4) based on subsequent diabetes occurrence, and iii) newly diagnosed T2D (n = 9). We found that impaired glucose tolerance associated with a global increase of plasma circulating microRNAs. While miR-148 and miR-222 were specifically modulated in diabetic subjects and correlated with parameters of glucose tolerance, the most accentuated microRNA dysregulation was found in NP IGT subjects, with increased level of miR-122, miR-99 and decreased level of let-7d, miR-18a, miR-18b, miR-23a, miR-27a, miR-28 and miR-30d in comparison with either NGT or T2D. Interestingly, several of these microRNAs significantly correlated with parameters of cholesterol metabolism. In conclusion, we observed the major perturbation of plasma circulating microRNA in NP pre-diabetic subjects and identified a unique microRNA profile that may become helpful in predicting diabetic development.

The expression of the BPIFB4 and CXCR4 associates with sustained health in long-living individuals from Cilento-Italy.

The study of the health status in long-living individuals (LLIs) may help identifying health-span and life-span determinants. BPI-Fold-Containing-Family-B-Member-4 (BPIFB4) protein is higher in healthy vs. non-healthy (frail) LLIs serum and its longevity-associated variant forced expression improves cardiovascular outcomes in ischemia mice models. Thus, we tested the association of BPIFB4 and ischemia-responding HIF-1α pathway components (i.e. CXCR4, AK3, ALDO-C, ADM, VEGF-A, GLUT-1 and miR-210) with human life-span and health-span by analyzing mRNA expression in circulating mononuclear cells (MNCs) of LLIs (N=14 healthy; N=31 frail) and young controls (N=63).ALDO-C, ADM, VEGF-A and GLUT-1 significantly decreased and miR-210 increased in LLIs vs. CONTROLS: Only VEGF-A and GLUT-1 showed further significant reduction in healthy-LLIs vs. frail-LLIs comparison. While BPIFB4 and CXCR4 were similar between LLIs and controls, BPIFB4 was significantly higher and CXCR4 lower in healthy- versus frail-LLIs. On a new set of LLIs (N=7 healthy and N=5 non-healthy) we assessed a potentially correlated function with low CXCR4 expression. Healthy donors' MNCs showed efficient migration ability toward CXCR4 ligand SDF-1α/CXCL12 and high percentage of migrated CXCR4pos cells which inversely correlated with CXCR4 RNA expression. In conclusion, BPIFB4 and CXCR4 expression classify LLIs health status that correlates with maintained MNCs migration.

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.

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.

3D hydrogel environment rejuvenates aged pericytes for skeletal muscle tissue engineering.

Skeletal muscle tissue engineering is a promising approach for the treatment of muscular disorders. However, the complex organization of muscle, combined with the difficulty in finding an appropriate source of regenerative cells and in providing an adequate blood supply to the engineered tissue, makes this a hard task to face. In the present work, we describe an innovative approach to rejuvenate adult skeletal muscle-derived pericytes (MP) based on the use of a PEG-based hydrogel scaffold. MP were isolated from young (piglet) and adult (boar) pigs to assess whether aging affects tissue regeneration efficiency. In vitro, MP from boars had similar morphology and colony forming capacity to piglet MP, but an impaired ability to form myotubes and capillary-like structures. However, the use of a PEG-based hydrogel to support adult MP significantly improved their myogenic differentiation and angiogenic potentials in vitro and in vivo. Thus, PEG-based hydrogel scaffolds may provide a progenitor cell "niche" that promotes skeletal muscle regeneration and blood vessel growth, and together with pericytes may be developed for use in regenerative applications.

Migratory activity of circulating mononuclear cells is associated with cardiovascular mortality in type 2 diabetic patients with critical limb ischemia.

OBJECTIVE: Prediction of clinical outcome in diabetic patients with critical limb ischemia (CLI) is unsatisfactory. This prospective study investigates if the abundance and migratory activity of a subpopulation of circulating mononuclear cells, namely, CD45(dim)CD34(pos)CXCR4(pos)KDR(pos) cells, predict major amputation and cardiovascular death in type 2 diabetic patients undergoing percutaneous transluminal angioplasty for CLI. RESEARCH DESIGN AND METHODS: A consecutive series of 119 type 2 diabetic patients with CLI was enrolled. CD45(dim)CD34(pos)CXCR4(pos)KDR(pos) cells were assessed by flow cytometry upon isolation and also after spontaneous or stromal cell-derived factor 1α-directed migration in an in vitro assay. The association between basal cell counts and migratory activity and the risk of an event at 18-month follow-up was evaluated in a multivariable regression analysis. RESULTS: Time-to-event analysis of amputation (n = 13) showed no association with the candidate predictors. Sixteen cardiovascular deaths occurred during 18 months of follow-up. Abundance of CD45(dim)CD34(pos)CXCR4(pos)KDR(pos) cells was not associated with cardiovascular mortality. Interestingly, in vitro migration of CD45(dim)CD34(pos)CXCR4(pos)KDR(pos) cells was higher in patients with cardiovascular death compared with event-free subjects (percentage of migrated cells median value and interquartile range, 0.03 [0.02-0.07] vs. 0.01 [0.01-0.03]; P = 0.0095). Multivariable regression model analysis showed that cell migration forecasts cardiovascular mortality independently of other validated predictors, such as age, diagnosed coronary artery disease, serum C-reactive protein, and estimated glomerular filtration rate. In this model, doubling of migrated cell counts increases the cardiovascular death hazard by 100% (P < 0.0001). CONCLUSIONS: The new predictor could aid in the identification of high-risk patients with type 2 diabetes requiring special diagnostic and therapeutic care after revascularization.