Publications by authors named "Paolo Madeddu"

172 Publications

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

Front Cardiovasc Med 2020 21;7:609980. Epub 2021 Jan 21.

Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol, United Kingdom.

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.
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http://dx.doi.org/10.3389/fcvm.2020.609980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859275PMC
January 2021

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

Free Radic Biol Med 2021 Jan 23;165:137-151. Epub 2021 Jan 23.

Bristol Medical School (Translational Health Sciences), Bristol Heart Institute, University of Bristol, Bristol, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.01.029DOI Listing
January 2021

Cell therapy for the treatment of heart disease: Renovation work on the broken heart is still in progress.

Authors:
Paolo Madeddu

Free Radic Biol Med 2021 Feb 6;164:206-222. Epub 2021 Jan 6.

Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol Royal Infirmary, Upper Maudlin Street, BS28HW, Bristol, United Kingdom. Electronic address:

Cardiovascular disease (CVD) continues to be the number one killer in the aging population. Heart failure (HF) is also an important cause of morbidity and mortality in patients with congenital heart disease (CHD). Novel therapeutic approaches that could restore stable heart function are much needed in both paediatric and adult patients. Regenerative medicine holds promises to provide definitive solutions for correction of congenital and acquired cardiac defects. In this review article, we recap some important aspects of cardiovascular cell therapy. First, we report quantifiable data regarding the scientific advancements in the field and how this has been translated into tangible outcomes according clinical studies and related meta-analyses. We then comment on emerging trends and technologies, such as the use of second-generation cell products, including pericyte-like vascular progenitors, and reprogramming of cells by different approaches including modulation of oxidative stress. The more affordable and feasible strategy of repurposing clinically available drugs to awaken the intrinsic healing potential of the heart will be discussed in the light of current social, financial, and ethical context. Cell therapy remains a work in progress field. Uncertainty in the ability of the experts and policy makers to solve urgent medical problems is growing in a world that is significantly influenced by them. This is particularly true in the field of regenerative medicine, due to great public expectations, polarization of leadership and funding, and insufficient translational vision. Cardiovascular regenerative medicine should be contextualized in a holistic program with defined priorities to allow a complete realization. Reshaping the notion of medical expertise is fundamental to fill the current gap in translation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.12.444DOI Listing
February 2021

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

Front Cardiovasc Med 2020 19;7:598890. Epub 2020 Nov 19.

Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol, United Kingdom.

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.
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http://dx.doi.org/10.3389/fcvm.2020.598890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711071PMC
November 2020

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

Antioxid Redox Signal 2020 Nov 23. Epub 2020 Nov 23.

Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol, United Kingdom.

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. 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. This study newly indicates that pericytes are a source of the matricellular protein SPARC. Modulation of SPARC production by pericytes may have potential implications for postinfarct healing.
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http://dx.doi.org/10.1089/ars.2019.7969DOI Listing
November 2020

Defective Cardiomyocyte Maturation in the Congenitally Hypoplastic Right Ventricle: A Wrong Molecular Trajectory Leading to Heart Failure?

Authors:
Paolo Madeddu

J Am Heart Assoc 2020 10 16;9(20):e019433. Epub 2020 Oct 16.

University of Bristol United Kingdom.

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http://dx.doi.org/10.1161/JAHA.120.019433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763371PMC
October 2020

Molecular therapies delaying cardiovascular aging: disease- or health-oriented approaches.

Vasc Biol 2020 16;2(1):R45-R58. Epub 2020 Jan 16.

Bristol Medical School (Translational Health Sciences), Bristol Heart Institute, University of Bristol, Bristol, UK.

Regenerative medicine is a new therapeutic modality that aims to mend tissue damage by encouraging the reconstitution of physiological integrity. It represents an advancement over conventional therapies that allow reducing the damage but result in disease chronicization. Age-related decline in spontaneous capacity of repair, especially in organs like the heart that have very limited proliferative capacity, contributes in reducing the benefit of conventional therapy. ncRNAs are emerging as key epigenetic regulators of cardiovascular regeneration. Inhibition or replacement of miRNAs may offer reparative solutions to cardiovascular disease. The first part of this review article is devoted to illustrating novel therapies emerging from research on miRNAs. In the second part, we develop new therapeutic concepts emerging from genetics of longevity. Prolonged survival, as in supercentenarians, denotes an exceptional capacity to repair and cope with risk factors and diseases. These characteristics are shared with offspring, suggesting that the regenerative phenotype is heritable. New evidence indicates that genetic traits responsible for prolongation of health span in humans can be passed to and benefit the outcomes of animal models of cardiovascular disease. Genetic studies have also focused on determinants of accelerated senescence and related druggable targets. Evolutionary genetics assessing the genetic basis of adaptation and comparing successful and unsuccessful genetic changes in response to selection within populations represent a powerful basis to develop novel therapies aiming to prolong cardiovascular and whole organism health.
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http://dx.doi.org/10.1530/VB-19-0029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439942PMC
January 2020

Shifting to a circular approach in publishing research data: new opportunities to develop and promote ideas and curricula in vascular biology.

Authors:
Paolo Madeddu

Vasc Biol 2020 15;2(1):E7-E9. Epub 2020 Jun 15.

University of Bristol, Bristol, UK.

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http://dx.doi.org/10.1530/VB-20-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439837PMC
June 2020

Cardiovascular complications of COVID-19: evidence, misconceptions, and new opportunities.

Authors:
Paolo Madeddu

Vasc Biol 2020 8;2(1):E3-E6. Epub 2020 Jun 8.

Bristol Medical School, Translational Health Sciences, University of Bristol, Bristol, UK.

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http://dx.doi.org/10.1530/VB-20-0008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439917PMC
June 2020

COVID-19, State of the Adult and Pediatric Heart: From Myocardial Injury to Cardiac Effect of Potential Therapeutic Intervention.

Front Cardiovasc Med 2020 14;7:140. Epub 2020 Jul 14.

Bristol Heart Institute, Translational Health Sciences, Bristol Royal Infirmary, University of Bristol, Bristol, United Kingdom.

While the COVID-19 pandemic continues to spread rapidly, resulting in considerable morbidity and mortality worldwide, multiple efforts are being made by the international scientific community to understand the pathogenesis of the viral infection and its clinical outcome. Older age and comorbidities have consistently been reported as risk factors for unfavorable prognosis, with cardiovascular disease accounting for up to 10 % of comorbid conditions among the infected patients. An understanding of the mechanism underlying the effect of this infection on patients with cardiovascular disease is essential to manage and improve clinical strategies against the disease in that population. In this review, we summarize the impact of COVID-19 on patients with underlying cardiovascular conditions and the cardiac implications of known and emerging therapeutic strategies. Our future effort will aim to further elucidate how the type and severity of the cardiac disease, with particular regard to Congenital Heart Disease, influences the prognosis and the outcome of the viral infection.
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http://dx.doi.org/10.3389/fcvm.2020.00140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371783PMC
July 2020

A Critical Role for Perivascular Cells in Amplifying Vascular Leakage Induced by Dengue Virus Nonstructural Protein 1.

mSphere 2020 08 5;5(4). Epub 2020 Aug 5.

Department of Biochemical Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom

Dengue is the most prevalent arthropod-borne viral disease affecting humans, with severe dengue typified by potentially fatal microvascular leakage and hypovolemic shock. Blood vessels of the microvasculature are composed of a tubular structure of endothelial cells ensheathed by perivascular cells (pericytes). Pericytes support endothelial cell barrier formation and maintenance through paracrine and contact-mediated signaling and are critical to microvascular integrity. Pericyte dysfunction has been linked to vascular leakage in noncommunicable pathologies such as diabetic retinopathy but has never been linked to infection-related vascular leakage. Dengue vascular leakage has been shown to result in part from the direct action of the secreted dengue virus (DENV) nonstructural protein NS1 on endothelial cells. Using primary human vascular cells, we show here that NS1 also causes pericyte dysfunction and that NS1-induced endothelial hyperpermeability is more pronounced in the presence of pericytes. Notably, NS1 specifically disrupted the ability of pericytes to support endothelial cell function in a three-dimensional (3D) microvascular assay, with no effect on pericyte viability or physiology. These effects are mediated at least in part through contact-independent paracrine signals involved in endothelial barrier maintenance by pericytes. We therefore identify a role for pericytes in amplifying NS1-induced microvascular hyperpermeability in severe dengue and thus show that pericytes can play a critical role in the etiology of an infectious vascular leakage syndrome. These findings open new avenues of research for the development of drugs and diagnostic assays for combating infection-induced vascular leakage, such as severe dengue. The World Health Organization considers dengue one of the top 10 global public health problems. There is no specific antiviral therapy to treat dengue virus and no way of predicting which patients will develop potentially fatal severe dengue, typified by vascular leakage and circulatory shock. We show here that perivascular cells (pericytes) amplify the vascular leakage-inducing effects of the dengue viral protein NS1 through contact-independent signaling to endothelial cells. While pericytes are known to contribute to noncommunicable vascular leakage, this is the first time these cells have been implicated in the vascular effects of an infectious disease. Our findings could pave the way for new therapies and diagnostics to combat dengue and potentially other infectious vascular leakage syndromes.
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http://dx.doi.org/10.1128/mSphere.00258-20DOI Listing
August 2020

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

Diabetologia 2020 10 30;63(10):2205-2217. Epub 2020 Jul 30.

Bristol Medical School, Translational Health Sciences, University of Bristol, Upper Maudlin Street, Bristol, BS2 8HW, UK.

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.
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http://dx.doi.org/10.1007/s00125-020-05230-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476972PMC
October 2020

Wharton's Jelly-Mesenchymal Stem Cell-Engineered Conduit for Pediatric Translation in Heart Defect.

Tissue Eng Part A 2021 Feb 31;27(3-4):201-213. Epub 2020 Jul 31.

Bristol Heart Institute, Translational Health Sciences, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom.

The materials available for the right ventricular outflow tract (RVOT) reconstruction in patients with tetralogy of fallot (TOF)/pulmonary atresia come with the severe limitation of long-term degeneration and lack of growth potential, causing right ventricular dysfunction, aneurysm formation, and arrhythmias, thus necessitating several high-risk reoperations throughout patients' lives. In this study, we evaluated the capacity of mesenchymal stem cells (MSCs) derived from the Wharton's Jelly (WJ-MSCs), the gelatinous inner portion of the umbilical cord, to grow and recellularize an extracellular matrix (ECM) graft in our optimized xeno-free, good manufacturing practice-compliant culture system. WJ-MSCs were phenotypically and functionally characterized by flow cytometry and multilineage differentiation capacity, respectively. The typical MSC immunophenotype and functional characteristics were retained in our xeno-free culture system, as well as the capacity to grow and engraft onto a naturally occurring scaffold. WJ-MSCs, from both human and swine source, showed excellent capacity to recellularize ECM graft producing a living cell-seeded construct. In addition, we have provided an proof of concept of feasibility of the cellularized conduit, engineered with swine WJ-MSCs, to be used in a novel porcine model of main pulmonary artery reconstruction, where it showed good integration within the host tissue. Our study indicates that the addition of WJ-MSCs to the ECM scaffold can upgrade the material, converting it into a living tissue, with the potential to grow, repair, and remodel the RVOT. These results could potentially represent a paradigm shift in pediatric cardiac intervention toward new modalities for effective and personalized surgical restoration of pulmonary artery and RVOT function in TOF/pulmonary atresia patients. Impact Statement The materials available for pulmonary artery reconstruction in pediatric patients with Congenital Heart Defect come with the limitation of long-term degeneration and lack of growth, thus necessitating several reoperations. Here, we describe a novel approach combining perinatal stem cells and naturally occurring scaffold to create a living tissue engineered conduit that showed good growth potential in a pulmonary artery reconstruction porcine model. We envision this approach is of great interest and relevance in tissue engineering field applied to cardiovascular regenerative medicine, as it may open up new avenues for correction of congenital cardiac defects, with remarkable medical and social benefits.
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http://dx.doi.org/10.1089/ten.TEA.2020.0088DOI Listing
February 2021

Circulating BPIFB4 Levels Associate With and Influence the Abundance of Reparative Monocytes and Macrophages in Long Living Individuals.

Front Immunol 2020 29;11:1034. Epub 2020 May 29.

Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, Salerno, Italy.

Long-Living Individuals (LLIs) delay aging and are less prone to chronic inflammatory reactions. Whether a distinct monocytes and macrophages repertoire is involved in such a characteristic remains unknown. Previous studies from our group have shown high levels of the host defense BPI Fold Containing Family B Member 4 (BPIFB4) protein in the peripheral blood of LLIs. Moreover, a polymorphic variant of the gene associated with exceptional longevity () confers protection from cardiovascular diseases underpinned by low-grade chronic inflammation, such as atherosclerosis. We hypothesize that BPIFB4 may influence monocytes pool and macrophages skewing, shifting the balance toward an anti-inflammatory phenotype. We profiled circulating monocytes in 52 LLIs (median-age 97) and 52 healthy volunteers (median-age 55) using flow cytometry. If the frequency of total monocyte did not change, the intermediate CD14++CD16+ monocytes counts were lower in LLIs compared to control adults. Conversely, non-classical CD14+CD16++ monocyte counts, which are M2 macrophage precursors with an immunomodulatory function, were found significantly associated with the LLIs' state. In a differentiation assay, supplementation of the LLIs' plasma enhanced the capacity of monocytes, either from LLIs or controls, to acquire a paracrine M2 phenotype. A neutralizing antibody against the phosphorylation site (ser 75) of BPIFB4 blunted the M2 skewing effect of the LLIs' plasma. These data indicate that LLIs carry a peculiar anti-inflammatory myeloid profile, which is associated with and possibly sustained by high circulating levels of BPIFB4. Supplementation of recombinant BPIFB4 may represent a novel means to attenuate inflammation-related conditions typical of unhealthy aging.
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http://dx.doi.org/10.3389/fimmu.2020.01034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272600PMC
May 2020

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

Cells 2020 05 21;9(5). Epub 2020 May 21.

Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol BS2 8HW UK.

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 gene protects cardiac function in the mouse model. This study aimed to determine the effect of therapy on the metabolic phenotype (ultra-high-performance liquid chromatography-mass spectrometry, UHPLC-MS) and cardiac transcriptome (next-generation RNAseq) in 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 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 - and vehicle-treated diabetic hearts revealed eight genes differentially expressed, mainly associated with mitochondrial and metabolic function. Bioinformatic analysis indicated that 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 -induced cardio-protection in a murine model of type-2 diabetes.
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http://dx.doi.org/10.3390/cells9051283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290798PMC
May 2020

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

Front Endocrinol (Lausanne) 2020 23;11:254. Epub 2020 Apr 23.

IRCCS MultiMedica, Milan, Italy.

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.
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http://dx.doi.org/10.3389/fendo.2020.00254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192007PMC
April 2020

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.

Eur J Heart Fail 2020 09 8;22(9):1568-1581. Epub 2020 May 8.

Translational Health Sciences, Bristol Medical School, University of Bristol, Bristol, UK.

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.
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http://dx.doi.org/10.1002/ejhf.1840DOI Listing
September 2020

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.

Diabetes Care 2020 07 1;43(7):1520-1529. Epub 2020 May 1.

University of Bristol, Bristol, U.K.

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.
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http://dx.doi.org/10.2337/dc19-2227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305013PMC
July 2020

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

Clin Sci (Lond) 2020 04;134(8):1031-1048

Bristol Medical School (Translational Health Sciences), Bristol Heart Institute, University of Bristol, Upper Maudlin Street, Bristol BS2 8HW, U.K.

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.
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http://dx.doi.org/10.1042/CS20200220DOI Listing
April 2020

Angiogenesis in Tissue Engineering: As Nature Intended?

Front Bioeng Biotechnol 2020 20;8:188. Epub 2020 Mar 20.

Section of Cardiovascular Sciences, Department of Biochemical Sciences, University of Surrey, Guildford, United Kingdom.

Despite the steady increase in the number of studies focusing on the development of tissue engineered constructs, solutions delivered to the clinic are still limited. Specifically, the lack of mature and functional vasculature greatly limits the size and complexity of vascular scaffold models. If tissue engineering aims to replace large portions of tissue with the intention of repairing significant defects, a more thorough understanding of the mechanisms and players regulating the angiogenic process is required in the field. This review will present the current material and technological advancements addressing the imperfect formation of mature blood vessels within tissue engineered structures.
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http://dx.doi.org/10.3389/fbioe.2020.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099606PMC
March 2020

Coronary artery mechanics induces human saphenous vein remodelling recruitment of adventitial myofibroblast-like cells mediated by Thrombospondin-1.

Theranostics 2020 3;10(6):2597-2611. Epub 2020 Feb 3.

Unità di Ingegneria Tissutale Cardiovascolare; Centro Cardiologico Monzino, IRCCS; Milan, Italy.

: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for coronary bypass grafting (CABG), especially in multi-vessel coronary artery disease (CAD). The objective of the present work was to address the role of mechanical forces in the activation of maladaptive vein bypass remodeling, a process determining progressive occlusion and recurrence of ischemic heart disease. : We employed a custom bioreactor to mimic the coronary shear and wall mechanics in human SV vascular conduits and reproduce experimentally the biomechanical conditions of coronary grafting and analyzed vein remodeling process by histology, histochemistry and immunofluorescence. We also subjected vein-derived cells to cyclic uniaxial mechanical stimulation in culture, followed by phenotypic and molecular characterization using RNA and proteomic methods. We finally validated our results and using a model of SV carotid interposition in pigs. : Exposure to pulsatile flow determined a remodeling process of the vascular wall involving reduction in media thickness. Smooth muscle cells (SMCs) underwent conversion from contractile to synthetic phenotype. A time-dependent increase in proliferating cells expressing mesenchymal (CD44) and early SMC (SM22α) markers, apparently recruited from the SV adventitia, was observed especially in CABG-stimulated vessels. Mechanically stimulated SMCs underwent transition from contractile to synthetic phenotype. MALDI-TOF-based secretome analysis revealed a consistent release of Thrombospondin-1 (TSP-1), a matricellular protein involved in TGF-β-dependent signaling. TSP-1 had a direct chemotactic effect on SV adventitia resident progenitors (SVPs); this effects was inhibited by blocking TSP-1 receptor CD47. The involvement of TSP-1 in adventitial progenitor cells differentiation and graft intima hyperplasia was finally contextualized in the TGF-β-dependent pathway, and validated in a saphenous vein into carotid interposition pig model. : Our results provide the evidence of a matricellular mechanism involved in the human vein arterialization process controlled by alterations in tissue mechanics, and open the way to novel potential strategies to block VGD progression based on targeting cell mechanosensing-related effectors.
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http://dx.doi.org/10.7150/thno.40595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052885PMC
February 2020

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

J Am Heart Assoc 2020 02 11;9(4):e014214. Epub 2020 Feb 11.

Bristol Heart Institute Translational Health Sciences University of Bristol Bristol Royal Infirmary Bristol United Kingdom.

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.
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http://dx.doi.org/10.1161/JAHA.119.014214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070228PMC
February 2020

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

Diabetes Metab Res Rev 2020 03 15;36 Suppl 1:e3240. Epub 2019 Dec 15.

Bristol Heart Institute, Translational Health Sciences, University of Bristol, Bristol Royal Infirmary, Bristol, UK.

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.
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http://dx.doi.org/10.1002/dmrr.3240DOI Listing
March 2020

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

Front Cardiovasc Med 2019 27;6:177. Epub 2019 Nov 27.

Laboratory of Cardiovascular Research, IRCCS MultiMedica, Milan, Italy.

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.
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http://dx.doi.org/10.3389/fcvm.2019.00177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890607PMC
November 2019

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

Arterioscler Thromb Vasc Biol 2020 01 12;40(1):34-44. Epub 2019 Sep 12.

Experimental Cardiovascular Medicine, University of Bristol, United Kingdom (M.S., P.M.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.119.312863DOI Listing
January 2020

LAV-BPIFB4 associates with reduced frailty in humans and its transfer prevents frailty progression in old mice.

Aging (Albany NY) 2019 08 28;11(16):6555-6568. Epub 2019 Aug 28.

IRCCS Multimedica, Cardiovascular Department, Milan, Italy.

Background: There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, gene transfer delayed frailty progression.

Conclusions: These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.
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http://dx.doi.org/10.18632/aging.102209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738439PMC
August 2019

Successful Reconstruction of the Right Ventricular Outflow Tract by Implantation of Thymus Stem Cell Engineered Graft in Growing Swine.

JACC Basic Transl Sci 2019 Jun 24;4(3):364-384. Epub 2019 Jun 24.

University of Bristol, Bristol Heart Institute, Bristol, United Kingdom.

Graft cellularization holds great promise in overcoming the limitations associated with prosthetic materials currently used in corrective cardiac surgery. In this study, the authors evaluated the advantages of graft cellularization for right ventricular outflow tract reconstruction in a novel porcine model. After 4.5 months from implantation, improved myocardial strain, better endothelialization and cardiomyocyte incorporation, and reduced fibrosis were observed in the cellularized grafts compared with the acellular grafts. To the authors' knowledge, this is the first demonstration of successful right ventricular outflow tract correction using bioengineered grafts in a large animal model.
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http://dx.doi.org/10.1016/j.jacbts.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609916PMC
June 2019

Single systemic transfer of a human gene associated with exceptional longevity halts the progression of atherosclerosis and inflammation in ApoE knockout mice through a CXCR4-mediated mechanism.

Eur Heart J 2020 Jul;41(26):2487-2497

Department of Medicine, Surgery and Dentistry, "Scuola Medica Salernitana" University of Salerno, Via S. Allende, 84081 Baronissi (SA), Italy.

Aims: Here, we aimed to determine the therapeutic effect of longevity-associated variant (LAV)-BPIFB4 gene therapy on atherosclerosis.

Methods And Results: ApoE knockout mice (ApoE-/-) fed a high-fat diet were randomly allocated to receive LAV-BPIFB4, wild-type (WT)-BPIFB4, or empty vector via adeno-associated viral vector injection. The primary endpoints of the study were to assess (i) vascular reactivity and (ii) atherosclerotic disease severity, by Echo-Doppler imaging, histology and ultrastructural analysis. Moreover, we assessed the capacity of the LAV-BPIFB4 protein to shift monocyte-derived macrophages of atherosclerotic mice and patients towards an anti-inflammatory phenotype. LAV-BPIFB4 gene therapy rescued endothelial function of mesenteric and femoral arteries from ApoE-/- mice; this effect was blunted by AMD3100, a CXC chemokine receptor type 4 (CXCR4) inhibitor. LAV-BPIFB4-treated mice showed a CXCR4-mediated shift in the balance between Ly6Chigh/Ly6Clow monocytes and M2/M1 macrophages, along with decreased T cell proliferation and elevated circulating levels of interleukins IL-23 and IL-27. In vitro conditioning with LAV-BPIFB4 protein of macrophages from atherosclerotic patients resulted in a CXCR4-dependent M2 polarization phenotype. Furthermore, LAV-BPIFB4 treatment of arteries explanted from atherosclerotic patients increased the release of atheroprotective IL-33, while inhibiting the release of pro-inflammatory IL-1β, inducing endothelial nitric oxide synthase phosphorylation and restoring endothelial function. Finally, significantly lower plasma BPIFB4 was detected in patients with pathological carotid stenosis (>25%) and intima media thickness >2 mm.

Conclusion: Transfer of the LAV of BPIFB4 reduces the atherogenic process and skews macrophages towards an M2-resolving phenotype through modulation of CXCR4, thus opening up novel therapeutic possibilities in cardiovascular disease.
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http://dx.doi.org/10.1093/eurheartj/ehz459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340354PMC
July 2020

Reconstruction of the pulmonary artery by a novel biodegradable conduit engineered with perinatal stem cell-derived vascular smooth muscle cells enables physiological vascular growth in a large animal model of congenital heart disease.

Biomaterials 2019 10 20;217:119284. Epub 2019 Jun 20.

Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, UK. Electronic address:

Lack of growth potential of available grafts represents a bottleneck in the correction of congenital heart defects. Here we used a swine small intestinal submucosa (SIS) graft functionalized with mesenchymal stem cell (MSC)-derived vascular smooth muscle cells (VSMCs), for replacement of the pulmonary artery in piglets. MSCs were expanded from human umbilical cord blood or new-born swine peripheral blood, seeded onto decellularized SIS grafts and conditioned in a bioreactor to differentiate into VSMCs. Results indicate the equivalence of generating grafts engineered with human or swine MSC-derived VSMCs. Next, we conducted a randomized, controlled study in piglets (12-15 kg), which had the left pulmonary artery reconstructed with swine VSMC-engineered or acellular conduit grafts. Piglets recovered well from surgery, with no casualty and similar growth rate in either group. After 6 months, grafted arteries had larger circumference in the cellular group (28.3 ± 2.3 vs 18.3 ± 2.1 mm, P < 0.001), but without evidence of aneurism formation. Immunohistochemistry showed engineered grafts were composed of homogeneous endothelium covered by multi-layered muscular media, whereas the acellular grafts exhibited a patchy endothelial cell layer and a thinner muscular layer. RESULTS: show the feasibility and efficacy of pulmonary artery reconstruction using clinically available grafts engineered with allogeneic VSMCs in growing swine.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658806PMC
October 2019