Jing Zhao

Background: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re‐implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells.Methods: Canine MSCs were derived from bone marrow and validated by measuring the expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of spleen focus forming virus, cytomegalovirus, elongation factor 1α and simian virus 40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin‐producing capacity of transduced primary cMSCs was assessed by measuring the concentration of C‐peptide produced.Results: Primary cMSC could be readily expanded in culture and efficiently transduced using lentiviral vectors encoding proinsulin. Increasing the multiplicity of infection from 3 to 20 led to an increase in C‐peptide secretion (from 1700 to 4000 pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability.Conclusions: The results of the present study suggest that optimised lentiviral transduction of the insulin gene into primary cMSCs renders these cells capable of secreting insulin over both the short‐ and long‐term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.

Late outgrowth endothelial progenitor cells (LO‐EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO‐EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO‐EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm². The interaction was not adhesion molecule‐dependent and not enhanced by EC activation. LO‐EPC expressed high levels of the VE‐Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO‐EPC due to cell retraction. LO‐EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO‐EPC in vitro and that LO‐EPC have properties consistent with their potential role in vascular repair.

Renal transplantation is potentially curative in renal failure, but long-term efficacy is limited by untreatable chronic rejection. Endothelial damage contributes to chronic rejection and is potentially repairable by circulating endothelial progenitor cells (EPC). The frequency and function of EPC are variably influenced by end-stage renal failure (ESRF). Here, we isolated and functionally characterized the late outgrowth EPC (LO-EPC) from ESRF patients to investigate their potential for endothelial repair. Patients with ESRF generated more LO-EPC colonies than healthy controls and had higher plasma levels of IL-1rα, IL-16, IL-6, MIF, VEGF, Prolactin, and PLGF. Patients' LO-EPC displayed normal endothelial cell morphology, increased secretion of PLGF, MCP-1, and IL-1β, and normal network formation in vitro and in vivo. They demonstrated decreased adhesion to extracellular matrix. Integrin gene profiles and protein expression were comparable in patients and healthy volunteers. In some patients, mesenchymal stem cells (MSC) were co-isolated and could be differentiated into adipocytes and osteocytes in vitro. This is the first study to characterize LO-EPC from patients with ESRF. Their behavior in vitro reflects the presence of elevated trophic factors; their ability to proliferate in vitro and angiogenic function makes them candidates for prevention of chronic rejection. Their impaired adhesion and the presence of MSC are areas for potential therapeutic intervention.

Chronic allograft vasculopathy (CAV) is a major cause of organ transplant failure that responds poorly to treatment. Endothelial activation, dysfunction and apoptosis contribute to CAV, whereas strategies for protecting endothelium and maximizing endothelial repair may diminish it. Late outgrowth endothelial progenitor cells (LO-EPC) can home to areas of injury and integrate into damaged vessels, implying a role in vascular repair; however, in an allograft, LO-EPC would be exposed to the hazardous microenvironment associated with transplant-related ischaemia reperfusion (I/R) injury and persistent inflammation. We evaluated the in vitro effect of I/R injury and the proinflammatory cytokine tumour necrosis factor (TNF)-α on LO-EPC phenotype and function. We show that LO-EPC are intrinsically more tolerant than mature EC to I/R injury induced apoptosis, maintaining their proliferative, migratory and network formation capacity. Under inflammatory conditions, LO-EPC were activated and released higher levels of inflammatory cytokines, upregulated adhesion molecule expression, and were more susceptible to apoptosis. Lentiviral vector-mediated overexpression of the protective gene A20 in LO-EPC maintained their angiogenic phenotype and function, and protected them against TNF-α-mediated apoptosis, reducing ICAM-1 expression and inflammatory cytokine secretion. Administration of ex vivo modified LO-EPC overexpressing A20 might effect vascular repair of damaged allografts and protect from CAV.

Rationale : Mutations in the bone morphogenetic protein type II receptor (BMPR-II) are responsible for the majority of cases of heritable pulmonary arterial hypertension (PAH). Mutations lead to reduced Smad1/5-driven expression of inhibitor of DNA binding protein 1 (Id1) and loss of the growth suppressive effects of BMPs. The impact of existing PAH therapies on BMP signaling is lacking. Objective : Because prostacyclin analogues are effective treatments for clinical PAH, we hypothesized that these agents enhance Smad1/Id1 signaling. Methods and Results : Iloprost alone induced Id1 expression in human pulmonary artery smooth muscle cells (PASMCs), an effect that was independent of Smad1/5 activation but dependent on a cAMP-responsive element in the Id1 promoter. In addition, iloprost and treprostinil enhanced BMP-induced phosphorylation of Smad1/5 and Id1 expression in a cAMP-dependent manner. The mechanism involved suppression of inhibitory Smad, Smad6. Furthermore, iloprost rescued the deficit in Smad1/5 phosphorylation and Id gene expression in PASMCs harboring mutations in BMPR-II and restored growth suppression to BMP4 in mutant PASMCs. We confirmed a critical role for Id1 in PASMC proliferation. Reduced expression of Id1 was observed in concentric intimal lesions of heritable PAH cases. In the monocrotaline rat model of PAH, associated with reduced BMPR-II expression, we confirmed that treprostinil inhibited smooth muscle cell proliferation and prevented progression of PAH while enhancing Smad1/5 phosphorylation and Id1 gene expression. Conclusions : Prostacyclin analogues enhance Id1 expression in vitro and in vivo and restore deficient BMP signaling in BMPR-II mutant PASMCs.

Natriuretic peptide receptor A (NPR-A) is the main physiological receptor for atrial natriuretic peptide (ANP). Maximal activation of NPR-A guanylyl cyclase (GC) requires ANP binding and ATP interaction with a putative cytoplasmic site. This study investigates the regulatory effect of ATP on GC-coupled NPR-A activity in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Cyclic GMP production and competitive inhibition of [(125)I]ANP(1-28) binding were performed in solubilized glomerular and papillary renal membranes. Here, we report that incubation of renal glomerular and papillary membranes with ATP induced a concentration-dependent increase in basal and ANP(1-28)-stimulated GC activity that was significantly greater in SHR than in age-matched WKY. ATPgammaS was more effective than ATP and induced a greater stimulation of cGMP production in SHR than in WKY. In contrast, in solubilized membranes ATP exerted an inhibitory role on basal and ANP(1-28)-induced GC activity, suggesting that an accessory protein is required for ATP-induced GC activation. ATP increases NPR-A affinity for ANP(1-28) and decreased B(max) in crude and solubilized membranes. Kinetic analysis of GC-coupled NPR-A revealed that ATP reduced the Km and increased the V(max), an effect that was greater in SHR. Our observations indicate that ATP exerts a greater net effect on NPR-A in SHR than in WKY, which might explain the greater rate of cGMP production observed in SHR compared to WKY.