Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplementation with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.

Zhou MS, Chadipiralla K, Mendez AJ, Jaimes EA, Silverstein RL, Webster K, Raij L. Nicotine potentiates proatherogenic effects of oxLDL by stimulating and upregulating macrophage CD36 signaling. Am J Physiol Heart Circ Physiol 305: H563-H574, 2013. First published June 7, 2013 doi:10.1152/ajpheart.00042.2013.-Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. CD36 mediates oxidized LDL (oxLDL) uptake and contributes to macrophage foam cell formation. We investigated a role for the CD36 pathway in nicotine-induced activation of macrophages and foam cell formation in vitro and in vivo. Nicotine in the same plasma concentration range found in smokers increased the CD36 ϩ /CD14 ϩ cell population in human peripheral blood mononuclear cells, increased CD36 expression of human THP1 macrophages, and increased macrophage production of reactive oxygen species, PKC␦ phosphorylation, and peroxisome proliferator-activated receptor-␥ (PPAR␥) expression. Nicotine-induced CD36 expression was suppressed by antioxidants and by specific PKC␦ and PPAR␥ inhibitors, implicating mechanistic roles for these intermediates. Nicotine synergized with oxLDL to increase macrophage expression of CD36 and cytokines TNF-␣, monocyte chemoattractant protein-1, IL-6, and CXCL9, all of which were prevented by CD36 small interfering (si)RNA. Incubation with oxLDL (50 g/ml) for 72 h resulted in lipid deposition in macrophages and foam cell formation. Preincubation with nicotine further increased oxLDL-induced lipid accumulation and foam cell formation, which was also prevented by CD36 siRNA. Treatment of apoE Ϫ/Ϫ mice with nicotine markedly exacerbated inflammatory monocyte levels and atherosclerotic plaque accumulation, effects that were not seen in CD36 Ϫ/Ϫ apoE Ϫ/Ϫ mice. Our results show that physiological levels of nicotine increase CD36 expression in macrophages, a pathway that may account at least in part for the known proinflammatory and proatherogenic properties of nicotine. These results identify such enhanced CD36 expression as a novel nicotine-mediated pathway that may constitute an independent risk factor for atherosclerosis in smokers. The results also suggest that exacerbated atherogenesis by this pathway may be an adverse side effect of extended use of high concentrations of nicotine independent of their mode of administration.nicotine; CD36; inflammatory cytokine; macrophage; foam cell SMOKING ACCOUNTS FOR 175,000 cardiovascular (CV) deaths annually in the US. The range of CV diseases exacerbated by cigarette smoke includes diabetic and obesity-related vascular diseases, stroke, myocardial infarction, peripheral vascular, and renal disease (20,35,50). Heart attacks are three times more common in smokers than in nonsmokers, and it is estimated that ϳ30% of deaths from coronary heart disease in the US are attributable to smoking (35). We and others (16,18) have demonstrated the importance of chemically stable compounds present in the gas phase of cigarette smoke in mediating endothelial injury and a...

Background: The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. Methods: Intrarenal expression of angiotensin II type 1 (AT₁R), type 2 (AT₂R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. Results: Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-β expression (7-fold). AT₁R /AT₂R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (–50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical –40%, medullary –53%) and medullary endothelial nitric oxide synthase (–48%) were decreased in old rats. Conclusion: Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly.

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet–induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1β and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2–4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet–induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

The c-Jun N-terminal kinases (JNKs) belong to the mitogen-activated protein kinases superfamily, which play an important role in the pathogenesis of cardiovascular and metabolic diseases. However, it is still unclear whether JNK participates in the regulation of vascular tone. We investigated the effect of JNK inhibitors on vascular reactivity in aortic rings in organ bath and on angiotensin (Ang) II-induced pressor responses in vivo in Sprague-Dawley (SD) rats. In aortic rings from SD rats, KCl, norepinephrine (NE), Ang II, or endothelin 1 (ET)-1 induced a dose-dependent vasoconstriction. Preincubation with the JNK inhibitor SP600125 (20 micromol/L) slightly inhibited KCl-induced vasoconstriction (Emax: -19%) and markedly inhibited vasoconstriction to NE (-42%), Ang II (-54%), and ET-1 (-42%). SP600125 induced a dose-dependent relaxation in the NE-preconstricted aortic rings (-54%) but exerted minimal relaxation in the KCI-preconstriction rings. To exclude the nonspecific effect of SP600125, we performed additional experiments using JNK peptide inhibitor 1, L-stereoisomer (L-JNKI1), a cell-permeable peptide inhibitor specific for JNK. Compared to SP600125, L-JNKI1 (20 micromol/L) had a smaller but still significant inhibitory effect on NE-induced vasoconstriction (-18%) and did not inhibit KCI-induced vasoconstriction. Next, we investigated the effect of L-JNKI1 (5 mg/kg intravenously [IV]) in vivo on Ang II-induced pressor responses in SD rats. Ang II induces a dose-dependent increase in systolic blood pressure and L-JNKI1 slightly attenuated the Ang II-induced pressor response. These results suggest that JNK signaling plays a role in the regulation of vascular tone.