The role of cardiac pericytes in health and disease: therapeutic targets for myocardial infarction.

Millions of cardiomyocytes die immediately after myocardial infarction, regardless of whether the culprit coronary artery undergoes prompt revascularization. Residual ischaemia in the peri-infarct border zone causes further cardiomyocyte damage, resulting in a progressive decline in contractile function. To date, no treatment has succeeded in increasing the vascularization of the infarcted heart. In the past decade, new approaches that can target the heart's highly plastic perivascular niche have been proposed. The perivascular environment is populated by mesenchymal progenitor cells, fibroblasts, myofibroblasts and pericytes, which can together mount a healing response to the ischaemic damage. In the infarcted heart, pericytes have crucial roles in angiogenesis, scar formation and stabilization, and control of the inflammatory response. Persistent ischaemia and accrual of age-related risk factors can lead to pericyte depletion and dysfunction. In this Review, we describe the phenotypic changes that characterize the response of cardiac pericytes to ischaemia and the potential of pericyte-based therapy for restoring the perivascular niche after myocardial infarction. Pericyte-related therapies that can salvage the area at risk of an ischaemic injury include exogenously administered pericytes, pericyte-derived exosomes, pericyte-engineered biomaterials, and pharmacological approaches that can stimulate the differentiation of constitutively resident pericytes towards an arteriogenic phenotype. Promising preclinical results from in vitro and in vivo studies indicate that pericytes have crucial roles in the treatment of coronary artery disease and the prevention of post-ischaemic heart failure.

The tyrosine kinase inhibitor Dasatinib reduces cardiac steatosis and fibrosis in obese, type 2 diabetic mice.

BACKGROUND: Cardiac steatosis is an early yet overlooked feature of diabetic cardiomyopathy. There is no available therapy to treat this condition. Tyrosine kinase inhibitors (TKIs) are used as first or second-line therapy in different types of cancer. In cancer patients with diabetes mellitus, TKIs reportedly improved glycemic control, allowing insulin discontinuation. They also reduced liver steatosis in a murine model of non-alcoholic fatty liver disease. The present study aimed to determine the therapeutic effect of the second-generation TKI Dasatinib on lipid accumulation and cardiac function in obese, type 2 diabetic mice. We also assessed if the drug impacts extra-cardiac fat tissue depots. METHODS: Two studies on 21-week-old male obese leptin receptor mutant BKS.Cg-+Leprdb/+Leprdb/OlaHsd (db/db) mice compared the effect of Dasatinib (5 mg/kg) and vehicle (10% DMSO + 90% PEG-300) given via gavage once every three days for a week or once every week for four weeks. Functional and volumetric indices were studied using echocardiography. Post-mortem analyses included the assessment of fat deposits and fibrosis using histology, and senescence using immunohistochemistry and flow cytometry. The anti-adipogenic action of Dasatinib was investigated on human bone marrow (BM)-derived mesenchymal stem cells (MSCs). Unpaired parametric or non-parametric tests were used to compare two and multiple groups as appropriate. RESULTS: Dasatinib reduced steatosis and fibrosis in the heart of diabetic mice. The drug also reduced BM adiposity but did not affect other fat depots. These structural changes were associated with improved diastolic indexes, specifically the E/A ratio and non-flow time. Moreover, Dasatinib-treated mice had lower levels of p16 in the heart compared with vehicle-treated controls, suggesting an inhibitory impact of the drug on the senescence signalling pathway. In vitro, Dasatinib inhibited human BM-MSC viability and adipogenesis commitment. CONCLUSIONS: Our findings suggest that Dasatinib opposes heart and BM adiposity and cardiac fibrosis. In the heart, this was associated with favourable functional consequences, namely improvement in an index of diastolic function. Repurposing TKI for cardiac benefit could address the unmet need of diabetic cardiac steatosis.

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice.

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein.

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.

Cardiac pericyte reprogramming by MEK inhibition promotes arteriologenesis and angiogenesis of the ischemic heart.

Pericytes (PCs) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated whether PCs can be reprogrammed to aid neovascularization. Primary PCs from human and mouse hearts acquired cytoskeletal proteins typical of vascular smooth muscle cells (VSMCs) upon exclusion of EGF/bFGF, which signal through ERK1/2, or upon exposure to the MEK inhibitor PD0325901. Differentiated PCs became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of 2 angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid-binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PCs during in vivo studies. In mice, implantation of Matrigel plugs containing human PCs plus PD0325901 promoted the formation of αSMA+ neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRß+AQP1+CRABP2+ PCs, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PCs have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.

The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.

Reconstruction of the Swine Pulmonary Artery Using a Graft Engineered With Syngeneic Cardiac Pericytes.

The neonatal heart represents an attractive source of regenerative cells. Here, we report the results of a randomized, controlled, investigator-blinded preclinical study, which assessed the safety and effectiveness of a matrix graft cellularized with cardiac pericytes (CPs) in a piglet model of pulmonary artery (PA) reconstruction. Within each of five trios formed by 4-week-old female littermate piglets, one element (the donor) was sacrificed to provide a source of CPs, while the other two elements (the graft recipients) were allowed to reach the age of 10 weeks. During this time interval, culture-expanded donor CPs were seeded onto swine small intestinal submucosa (SIS) grafts, which were then shaped into conduits and conditioned in a flow bioreactor. Control unseeded SIS conduits were subjected to the same procedure. Then, recipient piglets were randomized to surgical reconstruction of the left PA (LPA) with unseeded or CP-seeded SIS conduits. Doppler echocardiography and cardiac magnetic resonance imaging (CMRI) were performed at baseline and 4-months post-implantation. Vascular explants were examined using histology and immunohistochemistry. All animals completed the scheduled follow-up. No group difference was observed in baseline imaging data. The final Doppler assessment showed that the LPA's blood flow velocity was similar in the treatment groups. CMRI revealed a mismatch in the average growth of the grafted LPA and contralateral branch in both treatment groups. Histology of explanted arteries demonstrated that the CP-seeded grafts had a thicker luminal cell layer, more intraparietal arterioles, and a higher expression of endothelial nitric oxide synthase (eNOS) compared with unseeded grafts. Moreover, the LPA stump adjacent to the seeded graft contained more elastin and less collagen than the unseeded control. Syngeneic CP engineering did not accomplish the primary goal of supporting the graft's growth but was able to improve secondary outcomes, such as the luminal cellularization and intraparietal vascularization of the graft, and elastic remodeling of the recipient artery. The beneficial properties of neonatal CPs may be considered in future bioengineering applications aiming to reproduce the cellular composition of native arteries.

Treatment of COVID-19 by stage: any space left for mesenchymal stem cell therapy?

In many countries, COVID-19 now accounts for more deaths per year than car accidents and even the deadliest wars. Combating the viral pandemics requires a coordinated effort to develop therapeutic protocols adaptable to the disease severity. In this review article, we summarize a graded approach aiming to shield cells from SARS-CoV-2 entry and infection, inhibit excess inflammation and evasion of the immune response, and ultimately prevent systemic organ failure. Moreover, we focus on mesenchymal stem cell therapy, which has shown safety and efficacy as a treatment of inflammatory and immune diseases. The cell therapy approach is now repurposed in patients with severe COVID-19. Numerous trials of mesenchymal stem cell therapy are ongoing, especially in China and the USA. Leader companies in cell therapy have also started controlled trials utilizing their quality assessed cell products. Results are too premature to reach definitive conclusions.

Human adventitial pericytes provide a unique source of anti-calcific cells for cardiac valve engineering: Role of microRNA-132-3p.

AIMS: Tissue engineering aims to improve the longevity of prosthetic heart valves. However, the optimal cell source has yet to be determined. This study aimed to establish a mechanistic rationale supporting the suitability of human adventitial pericytes (APCs). METHODS AND RESULTS: APCs were immunomagnetically sorted from saphenous vein leftovers of patients undergoing coronary artery bypass graft surgery and antigenically characterized for purity. Unlike bone marrow-derived mesenchymal stromal cells (BM-MSCs), APCs were resistant to calcification and delayed osteochondrogenic differentiation upon high phosphate (HP) induction, as assessed by cytochemistry and expression of osteogenic markers. Moreover, glycolysis was activated during osteogenic differentiation of BM-MSCs, whereas APCs showed no increase in glycolysis upon HP challenge. The microRNA-132-3p (miR-132), a known inhibitor of osteogenesis, was found constitutively expressed by APCs and upregulated following HP stimulation. The anti-calcific role of miR-132 was further corroborated by in silico analysis, luciferase assays in HEK293 cells, and transfecting APCs with miR-132 agomir and antagomir, followed by assessment of osteochondrogenic markers. Interestingly, treatment of swine cardiac valves with APC-derived conditioned medium conferred them with resistance to HP-induced osteogenesis, with this effect being negated when using the medium of miR-132-silenced APCs. Additionally, as an initial bioengineering step, APCs were successfully engrafted onto pericardium sheets, where they proliferated and promoted aortic endothelial cells attraction, a process mimicking valve endothelialization. CONCLUSIONS: Human APCs are resistant to calcification compared with BM-MSCs and convey the anti-calcific phenotype to heart valves through miR-132. These findings may open new important avenues for prosthetic valve cellularization.

Heart failure impairs the mechanotransduction properties of human cardiac pericytes.

The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically.

Secreted Protein Acidic and Cysteine Rich Matricellular Protein is Enriched in the Bioactive Fraction of the Human Vascular Pericyte Secretome.

Aims: To ascertain if human pericytes produce SPARC (acronym for Secreted Protein Acidic and Cysteine Rich), a matricellular protein implicated in the regulation of cell proliferation, migration, and cell-matrix interactions; clarify if SPARC expression in cardiac pericytes is modulated by hypoxia; and determine the functional consequences of SPARC silencing. Results: Starting from the recognition that the conditioned media (CM) of human pericytes promote proliferation and migration of cardiac stromal cells, we screened candidate mediators by mass-spectrometry analysis. Of the 14 high-confidence proteins (<1% FDR) identified in the bioactive fractions of the pericyte CM, SPARC emerged as the top-scored matricellular protein. SPARC expression was validated using ELISA and found to be upregulated by hypoxia/starvation in pericytes that express platelet-derived growth factor receptor α (PDGFRα). This subfraction is acknowledged to play a key role in extracellular matrix remodeling. Studies in patients with acute myocardial infarction showed that peripheral blood SPARC correlates with the levels of creatine kinase Mb, a marker of cardiac damage. Immunohistochemistry analyses of infarcted hearts revealed that SPARC is expressed in vascular and interstitial cells. Silencing of SPARC reduced the pericyte ability to secrete collagen1a1, without inhibiting the effects of CM on cardiac and endothelial cells. These data indicate that SPARC is enriched in the bioactive fraction of the pericyte CM, is induced by hypoxia and ischemia, and is essential for pericyte ability to produce collagen. Innovation: This study newly indicates that pericytes are a source of the matricellular protein SPARC. Conclusion: Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.

Fabrication of New Hybrid Scaffolds for in vivo Perivascular Application to Treat Limb Ischemia.

Cell therapies are emerging as a new therapeutic frontier for the treatment of ischemic disease. However, femoral occlusions can be challenging environments for effective therapeutic cell delivery. In this study, cell-engineered hybrid scaffolds are implanted around the occluded femoral artery and the therapeutic benefit through the formation of new collateral arteries is investigated. First, it is reported the fabrication of different hybrid "hard-soft" 3D channel-shaped scaffolds comprising either poly(ε-caprolactone) (PCL) or polylactic-co-glycolic acid (PLGA) and electro-spun of gelatin (GL) nanofibers. Both PCL-GL and PLGA-GL scaffolds show anisotropic characteristics in mechanical tests and PLGA displays a greater rigidity and faster degradability in wet conditions. The resulting constructs are engineered using human adventitial pericytes (APCs) and both exhibit excellent biocompatibility. The 3D environment also induces expressional changes in APCs, conferring a more pronounced proangiogenic secretory profile. Bioprinting of alginate-pluronic gel (AG/PL), containing APCs and endothelial cells, completes the hybrid scaffold providing accurate spatial organization of the delivered cells. The scaffolds implantation around the mice occluded femoral artery shows that bioengineered PLGA hybrid scaffold outperforms the PCL counterpart accelerating limb blood flow recovery through the formation arterioles with diameters >50 µm, demonstrating the therapeutic potential in stimulating reparative angiogenesis.

Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF-1/CXCR4 signalling pathway.

AIMS: Homozygosity for a four-missense single-nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this longevity-associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. METHODS AND RESULTS: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild-type-BPIFB4 exerted no benefit. Interestingly, LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and up-regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 up-regulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. CONCLUSIONS: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Multi-Omics Analysis of Diabetic Heart Disease in the db/db Model Reveals Potential Targets for Treatment by a Longevity-Associated Gene.

Characterisation of animal models of diabetic cardiomyopathy may help unravel new molecular targets for therapy. Long-living individuals are protected from the adverse influence of diabetes on the heart, and the transfer of a longevity-associated variant (LAV) of the human BPIFB4 gene protects cardiac function in the db/db mouse model. This study aimed to determine the effect of LAV-BPIFB4 therapy on the metabolic phenotype (ultra-high-performance liquid chromatography-mass spectrometry, UHPLC-MS) and cardiac transcriptome (next-generation RNAseq) in db/db mice. UHPLC-MS showed that 493 cardiac metabolites were differentially modulated in diabetic compared with non-diabetic mice, mainly related to lipid metabolism. Moreover, only 3 out of 63 metabolites influenced by LAV-BPIFB4 therapy in diabetic hearts showed a reversion from the diabetic towards the non-diabetic phenotype. RNAseq showed 60 genes were differentially expressed in hearts of diabetic and non-diabetic mice. The contrast between LAV-BPIFB4- and vehicle-treated diabetic hearts revealed eight genes differentially expressed, mainly associated with mitochondrial and metabolic function. Bioinformatic analysis indicated that LAV-BPIFB4 re-programmed the heart transcriptome and metabolome rather than reverting it to a non-diabetic phenotype. Beside illustrating global metabolic and expressional changes in diabetic heart, our findings pinpoint subtle changes in mitochondrial-related proteins and lipid metabolism that could contribute to LAV-BPIFB4-induced cardio-protection in a murine model of type-2 diabetes.

In Vitro and In Vivo Preclinical Testing of Pericyte-Engineered Grafts for the Correction of Congenital Heart Defects.

Background We have previously reported the possibility of using pericytes from leftovers of palliative surgery of congenital heart disease to engineer clinically certified prosthetic grafts. Methods and Results Here, we assessed the feasibility of using prosthetic conduits engineered with neonatal swine pericytes to reconstruct the pulmonary artery of 9-week-old piglets. Human and swine cardiac pericytes were similar regarding anatomical localization in the heart and antigenic profile following isolation and culture expansion. Like human pericytes, the swine surrogates form clones after single-cell sorting, secrete angiogenic factors, and extracellular matrix proteins and support endothelial cell migration and network formation in vitro. Swine pericytes seeded or unseeded (control) CorMatrix conduits were cultured under static conditions for 5 days, then they were shaped into conduits and incubated in a flow bioreactor for 1 or 2 weeks. Immunohistological studies showed the viability and integration of pericytes in the outer layer of the conduit. Mechanical tests documented a reduction in stiffness and an increase in strain at maximum load in seeded conduits in comparison with unseeded conduits. Control and pericyte-engineered conduits were then used to replace the left pulmonary artery of piglets. After 4 months, anatomical and functional integration of the grafts was confirmed using Doppler echography, cardiac magnetic resonance imaging, and histology. Conclusions These findings demonstrate the feasibility of using neonatal cardiac pericytes for reconstruction of small-size branch pulmonary arteries in a large animal model.

Umbilical Cord Pericytes Provide a Viable Alternative to Mesenchymal Stem Cells for Neonatal Vascular Engineering.

Reconstructive surgery of congenital heart disease (CHD) remains inadequate due to the inability of prosthetic grafts to match the somatic growth of pediatric patients. Functionalization of grafts with mesenchymal stem cells (MSCs) may provide a solution. However, MSCs represent a heterogeneous population characterized by wide diversity across different tissue sources. Here we investigated the suitability of umbilical cord pericytes (UCPs) in neonatal vascular engineering. Explant outgrowth followed by immunomagnetic sorting was used to isolate neural/glial antigen 2 (NG2)+/CD31- UCPs. Expanded NG2 UCPs showed consistent antigenic phenotype, including expression of mesenchymal and stemness markers, and high proliferation rate. They could be induced to a vascular smooth muscle cell-like phenotype after exposure to differentiation medium, as evidenced by the expression of transgelin and smooth muscle myosin heavy chain. Analysis of cell monolayers and conditioned medium revealed production of extracellular matrix proteins and the secretion of major angiocrine factors, which conferred UCPs with ability to promote endothelial cell migration and tube formation. Decellularized swine-derived grafts were functionalized using UCPs and cultured under static and dynamic flow conditions. UCPs were observed to integrate into the outer layer of the graft and modify the extracellular environment, resulting in improved elasticity and rupture strain in comparison with acellular grafts. These findings demonstrate that a homogeneous pericyte-like population can be efficiently isolated and expanded from human cords and integrated in acellular grafts currently used for repair of CHD. Functional assays suggest that NG2 UCPs may represent a viable option for neonatal tissue engineering applications.

Personalized Cardiovascular Regenerative Medicine: Targeting the Extreme Stages of Life.

Cardiovascular regenerative medicine is an exciting new approach that promises to change the current care of million people world-wide. Major emphasis was given to the quality and quantities of regenerative products, but recent evidence points to the importance of a better specification of the target population that may take advantage of these advanced medical treatments. Patient stratification is an important step in drug development. Tailoring treatment to the patient's specificity allowed significant improvement in cancer therapy, but personalized regenerative medicine is still at the initial stage in the cardiovascular field. For example, new-borns with a congenital heart condition and elderly people require dedicated therapeutic approaches, which adapt to their lifetime needs. In these people, personalized treatments may overcome the benefits delivered by standard protocols. In this review, we provide insights into these extreme stages of life as potential targets for cardiovascular reconstitution.

Nerve growth factor gene therapy improves bone marrow sensory innervation and nociceptor-mediated stem cell release in a mouse model of type 1 diabetes with limb ischaemia.

AIMS/HYPOTHESIS: Sensory neuropathy is common in people with diabetes; neuropathy can also affect the bone marrow of individuals with type 2 diabetes. However, no information exists on the state of bone marrow sensory innervation in type 1 diabetes. Sensory neurons are trophically dependent on nerve growth factor (NGF) for their survival. The aim of this investigation was twofold: (1) to determine if sensory neuropathy affects the bone marrow in a mouse model of type 1 diabetes, with consequences for stem cell liberation after tissue injury; and (2) to verify if a single systemic injection of the NGF gene exerts long-term beneficial effects on these phenomena. METHODS: A mouse model of type 1 diabetes was generated in CD1 mice by administration of streptozotocin; vehicle was administered to non-diabetic control animals. Diabetic animals were randomised to receive systemic gene therapy with either human NGF or ß-galactosidase. After 13 weeks, limb ischaemia was induced in both groups to study the recovery post injury. When the animals were killed, samples of tissue and peripheral blood were taken to assess stem cell mobilisation and homing, levels of substance P and muscle vascularisation. An in vitro cellular model was adopted to verify signalling downstream to human NGF and related neurotrophic or pro-apoptotic effects. Normally distributed variables were compared between groups using the unpaired Student's t test and non-normally distributed variables were assessed by the Wilcoxon-Mann-Whitney test. The Fisher's exact test was employed for categorical variables. RESULTS: Immunohistochemistry indicated a 3.3-fold reduction in the number of substance P-positive nociceptive fibres in the bone marrow of type 1 diabetic mice (p < 0.001 vs non-diabetic). Moreover, diabetes abrogated the creation of a neurokinin gradient which, in non-diabetic mice, favoured the mobilisation and homing of bone-marrow-derived stem cells expressing the substance P receptor neurokinin 1 receptor (NK1R). Pre-emptive gene therapy with NGF prevented bone marrow denervation, contrasting with the inhibitory effect of diabetes on the mobilisation of NK1R-expressing stem cells, and restored blood flow recovery from limb ischaemia. In vitro hNGF induced neurite outgrowth and exerted anti-apoptotic actions on rat PC12 cells exposed to high glucose via activation of the canonical neurotrophic tyrosine kinase receptor type 1 (TrkA) signalling pathway. CONCLUSIONS/INTERPRETATION: This study shows, for the first time, the occurrence of sensory neuropathy in the bone marrow of type 1 diabetic mice, which translates into an altered modulation of substance P and depressed release of substance P-responsive stem cells following ischaemia. NGF therapy improves bone marrow sensory innervation, with benefits for healing on the occurrence of peripheral ischaemia. Nociceptors may represent a new target for the treatment of ischaemic complications in diabetes.