Induced pluripotent stem cells attenuate chronic allogeneic vasculopathy in an integrin beta-1-dependent manner.

This study aimed to determine the mechanism of isogeneic-induced pluripotent stem cells (iPSCs) homing to vascular transplants and their therapeutic effect on chronic allogeneic vasculopathy. We found that integrin ß1 (Intgß1) was the dominant integrin ß unit in iPSCs that mediates the adhesion of circulatory and endothelial cells (ECs). Intgß1 knockout or Intgß1-siRNAs inhibit iPSC adhesion and migration across activated endothelial monolayers. The therapeutic effects of the following were examined: iPSCs, Intgß1-knockout iPSCs, iPSCs transfected with Intgß1-siRNAs or nontargeting siRNAs, iPSC-derived ECs, iPSC-derived ECs simultaneously overexpressing Intgα4 and Intgß1, iPSCs precultured in endothelial medium for 3 days (endothelial-prone stem cells), primary aortic ECs, mouse embryonic fibroblasts, and phosphate-buffered saline (control). The cells were administered every 3 days for a period of 8 weeks. iPSCs, iPSCs transfected with nontargeting siRNAs, and endothelial-prone stem cells selectively homed on the luminal surface of the allografts, differentiated into ECs, and decreased neointimal proliferation. Through a single administration, we found that iPSCs trafficked to allograft lesions, differentiated into ECs within 1 week, and survived for 4-8 weeks. The therapeutic effect of a single administration was moderate. Thus, Intgß1 and pluripotency are essential for iPSCs to treat allogeneic vasculopathy.

Discovery and anti-inflammatory evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3ß (GSK-3ß).

Glycogen synthase kinase-3ß (GSK-3ß) has been identified to promote inflammation and its inhibitors have also been proven to treat some inflammatory mediated diseases in animal models. Non-ATP competitive inhibitors inherently have better therapeutical value due to their higher specificity than ATP competitive ones. In this paper, we designed and synthesized a series of new BTZ derivatives as non-ATP competitive GSK-3ß inhibitors. Kinetic analysis revealed two typical compounds 6j and 3j showed the different non-ATP competitive mechanism of substrate competition or allosteric modulation to GSK-3ß, respectively. As expected, the two compounds showed good specificity in a panel test of 16 protein kinases, even to the closest enzymes, like CDK-1/cyclin B and CK-II. The in vivo results proved that both compounds can greatly attenuate the LPS-induced acute lung injury (ALI) and diminish inflammation response in mice by inhibiting the mRNA expression of IL-1ß and IL-6. Western blot analysis demonstrated that they negatively regulated GSK-3ß, and the mechanism of the observed beneficial effects of the inhibitors may involve both the increased phosphorylation of the Ser9 residue on GSK-3ß and protein expression of Sirtuin 1 (SIRT1). The results support that such novel BTZ compounds have a protective role in LPS-induced ALI, and might be attractive candidates for further development of inflammation pharmacotherapy, which greatly thanks to their inherently high selectivities by the non-ATP competitive mode of action. Finally, we proposed suggesting binding modes by Docking study to well explain the impacts of compounds on the target site.

Nitric oxide-mediated the therapeutic properties of induced pluripotent stem cell for paraquat-induced acute lung injury.

The mortality rate associated with acute lung injury (ALI) and its severe form, acute respiratory distress syndrome, is high. Induced pluripotent stem cell (iPSC) therapy is a potential treatment method for ALI, but its therapeutic efficacy is limited in injured lungs. Nitric oxide (NO) has various physiological actions. The current study investigated the effect of iPSCs pretreated with NO donors in paraquat (PQ)-induced ALI mouse model. Male C57BL/6 mice were intraperitoneally injected with PQ, followed by infusion of phosphate-buffered saline, iPSCs, L-arginine pretreated iPSCs, or Nitro-L-arginine methylester (L-NAME) pretreated iPSCs through the tail veins. Histopathological changes, pulmonary microvascular permeability, and inflammatory cytokine levels were analyzed after 3 or 28 d. The effects on iPSC proliferation, migration, and adhesion were evaluated in vitro. More L-arginine-pretreated iPSCs were selectively trafficked into the injured pulmonary tissue of mice with LPS-induced ALI, drastically diminishing the histopathologic changes and inflammatory cytokine levels (IL-1ß and IL-6). There was also markedly improved pulmonary microvascular permeability and pulmonary function. The NO inhibitor abolished the protective effects of iPSCs. In addition, the ability of L-arginine to promote the proliferation and migration of iPSCs was decreased by L-NAME pretreatment, suggesting that NO might mediate the therapeutic benefits of iPSC. The improvement of the iPSC physiological changes by the endogenous gaseous molecule NO reduces lung injury severity. L-Arginine represents a pharmacologically important strategy for enhancing the therapeutic potential of iPSCs.

Enhanced wound healing promotion by immune response-free monkey autologous iPSCs and exosomes vs. their allogeneic counterparts.

BACKGROUND: Comparing non-inbred autologous and allogeneic induced pluripotent stem cells (iPSCs) and their secreted subcellular products among non-human primates is critical for choosing optimal iPSC products for human clinical trials. METHODS: iPSCs were induced from skin fibroblastic cells of adult male rhesus macaques belonging to four unrelated consanguineous families. Teratoma generativity, host immune response, and skin wound healing promotion were evaluated subsequently. FINDINGS: All autologous, but no allogeneic, iPSCs formed teratomas, whereas all allogeneic, but no autologous, iPSCs caused lymphocyte infiltration. Macrophages were not detectable in any wound. iPSCs expressed significantly more MAMU A and E of the major histocompatibility complex (MHC) class I but not more other MHC genetic alleles than parental fibroblastic cells. All topically disseminated autologous and allogeneic iPSCs, and their exosomes accelerated skin wound healing, as demonstrated by wound closure, epithelial coverage, collagen deposition, and angiogenesis. Allogeneic iPSCs and their exosomes were less effective and viable than their autologous counterparts. Some iPSCs differentiated into new endothelial cells and all iPSCs lost their pluripotency in 14 days. Exosomes increased cell viability of injured epidermal, endothelial, and fibroblastic cells in vitro. Although exosomes contained some mRNAs of pluripotent factors, they did not impart pluripotency to host cells. INTERPRETATION: Although all of the autologous and allogeneic iPSCs and exosomes accelerated wound healing, allogeneic iPSC exosomes were the preferred choice for "off-the shelf" iPSC products, owing to their mass-production, with no concern of teratoma formation. FUND: National Natural Science Foundation of China and National Key R&D Program of China.

VCAM-1-mediated neutrophil infiltration exacerbates ambient fine particle-induced lung injury.

BACKGROUND: Fine ambient particle matter (PM2.5) induces inflammatory lung injury; however, whether intratracheal administration of PM2.5 increases pulmonary polymorphonuclear leukocyte (PMN) infiltration, the mechanism of infiltration, and if these cells exacerbate PM2.5-induced lung injury are unknown. METHODS: Using 32,704 subjects, the association between blood PMNs and ambient PM2.5 levels on the previous day was retrospectively analyzed. Neutropenia was achieved by injecting mice with PMN-specific antibodies. Inhibition of PMN infiltration was achieved by pretreating PMNs with soluble vascular cell adhesion molecule-1 (sVCAM-1). The effects of PMNs on PM2.5-induced lung injury and endothelial dysfunction were observed. RESULT: Short-term PM2.5 (> 75 µg/m3 air) exposure increased the PMN/white blood cell ratio and the PMN count in human peripheral blood observed during routine examination. A significant number of PM2.5-treated PMNs was able to bind sVCAM-1. In mice, intratracheally-instilled PM2.5 deposited in the alveolar space and endothelial cells, which caused significant lung edema, morphological disorder, increased permeability of the endothelial-alveolar epithelial barrier, and PMN infiltration with increased VCAM-1 expression. Depletion of circulatory PMNs inhibited these adverse effects. Replenishment of untreated PMNs, but not those pretreated with soluble VCAM-1, restored lung injury. In vitro, PM2.5 increased VCAM-1 expression and endothelial and epithelial monolayer permeability, and promoted PMN adhesion to, chemotaxis toward, and migration across these monolayers. PMNs, but not those pretreated with soluble VCAM-1, exacerbated these effects. CONCLUSION: VCAM-1-mediated PMN infiltration was essential for a detrimental cycle of PM2.5-induced inflammation and lung injury. Results suggest that drugs that inhibit PMN function might prevent acute deterioration of chronic pulmonary and cardiovascular diseases triggered by PM2.5.

Direct in vivo application of induced pluripotent stem cells is feasible and can be safe.

Increasing evidence suggests the consensus that direct in vivo application of induced pluripotent stem cells (iPSCs) is infeasible may not be true. Methods: Teratoma formation and fate were examined in 53 normal and disease conditions involving brain, lung, liver, kidney, islet, skin, hind limb, and arteries. Results: Using classic teratoma generation assays, which require iPSCs to be congregated and confined, all mouse, human, and individualized autologous monkey iPSCs tested formed teratoma, while iPSC-derived cells did not. Intravenously or topically-disseminated iPSCs did not form teratomas with doses up to 2.5×108 iPSCs/kg and observation times up to 18 months, regardless of host tissue type; autologous, syngeneic, or immune-deficient host animals; presence or absence of disease; disease type; iPSC induction method; commercial or self-induced iPSCs; mouse, human, or monkey iPSCs; frequency of delivery; and sex. Matrigel-confined, but not PBS-suspended, syngeneic iPSCs delivered into the peritoneal cavity or renal capsule formed teratomas. Intravenously administered iPSCs were therapeutic with a dose as low as 5×106/kg and some iPSCs differentiated into somatic cells in injured organs. Disseminated iPSCs trafficked into injured tissue and survived significantly longer in injured than uninjured organs. In disease-free animals, no intravenously administered cell differentiated into an unwanted long-lasting cell or survived as a quiescent stem cell. In coculture, the stem cell medium and dominant cell-type status were critical for iPSCs to form cell masses. Conclusion: Teratoma can be easily and completely avoided by disseminating the cells. Direct in vivo iPSC application is feasible and can be safe.

Anti-serum with anti-autoantibody activity decreases autoantibody-positive B lymphocytes and type 1 diabetes of female NOD mice.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by an autoimmune-mediated loss of insulin secreting ß-cells. Each B lymphocyte clone that escapes immune tolerance produces a specific antibody. No specific treatment against autoantibodies is available for autoimmune diseases. We have developed a strategy to produce an antiserum against autoantibodies for the treatment of T1DM. Non-obese diabetic (NOD) but not Balb/c mouse serum contains autoantibodies. Antisera were produced by immunizing Balb/c mice with affinity-purified IgG from NOD or BALB/c mice along with the immune adjuvant (hereafter, NIgG or BIgG, respectively). A bolus administration of NIgG significantly reduced serum autoantibodies, autoantibody-positive B lymphocytes in the spleens of NOD mice, mortality and morbidity of diabetes, blood glucose and islet immune infiltration, whereas it increased islet mass in NOD mice for at least 26 weeks. NIgG antiserum treatment has no significant effect on CD3(+), CD4(+) or CD8(+) T cells and B220(+) or CD19(+) B cells. BIgG also imparted a moderate therapeutic effect, although it was considerably lower than that of NIgG. NIgG did not cross-react with allogeneic serum. NIgG showed no effect on Balb/c mice. The results show the feasibility of producing antiserum against autoantibodies to prevent and treat autoimmune-induced T1DM with a single bolus administration.