Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Polyetheretherketone (PEEK)/β‐tricalcium phosphate (β‐TCP) scaffolds are expected to be able to combine the excellent mechanical strength of PEEK and the good bioactivity and biodegradability of β‐TCP. While PEEK acts as a closed membrane in which β‐TCP is completely wrapped after the melting/solidifying processing, the PEEK membrane degrades very little, hence the scaffolds cannot display bioactivity and biodegradability. The strategy reported here is to blend a biodegradable polymer with PEEK and β‐TCP to fabricate multi‐material scaffolds via selective laser sintering (SLS). The biodegradable polymer first degrades and leaves caverns on the closed membrane, and then the wrapped β‐TCP is exposed to body fluid. In this study, poly(l‐lactide) (PLLA) is adopted as the biodegradable polymer. The results show that large numbers of caverns form on the membrane with the degradation of PLLA, enabling direct contact between β‐TCP and body fluid, and allowing for their ion‐exchange. As a consequence, the scaffolds display the bioactivity, biodegradability and cytocompatibility. Moreover, bone defect repair studies reveal that new bone tissues grow from the margin towards the center of the scaffolds from the histological analysis. The bone defect region is completely connected to the host bone end after 8 weeks of implantation.

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. ANIT toxicity toward mouse hepatocytes in vivo is mediated primarily by neutrophils via CD18. Am J Physiol Gastrointest Liver Physiol 291: G355-G363, 2006. First published April 13, 2006 doi:10.1152/ajpgi.00458.2005.-␣-Naphthylisothiocyanate (ANIT) is a hepatotoxicant that causes acute cholestatic hepatitis with infiltration of neutrophils around bile ducts and necrotic hepatocytes. The objective of this study was to determine whether the ␤2-integrin CD18, which plays an important role in leukocyte invasion and cytotoxicity, contributes to ANIT-induced hepatic inflammation and liver injury. Mice with varying levels of leukocyte CD18 expression were treated with ANIT and monitored for hepatic neutrophil influx and liver injury over 48 h. Mice that were partially deficient in CD18 (30% of normal levels) developed periportal inflammation and widespread hepatic necrosis after ANIT treatment in a pattern identical to that in wild-type (WT) mice. In contrast, mice that completely lack CD18 (CD18 null) were resistant to ANIT toxicity. Forty-eight hours after ANIT, CD18-null mice displayed 60% lower serum alanine aminotransferase (ALT) levels and 75% less hepatic necrosis, as shown by morphometry, than WT mice. This was true despite evidence that ANIT still provoked hepatic neutrophil influx in CD18-null mice. WT mice could also be protected from ANIT-induced hepatocellular necrosis, by depleting the animals of neutrophils. Notably, neither CD18-null mice nor neutrophil-depleted WT mice exhibited any attenuation of bile duct injury or cholestasis due to ANIT. We conclude from these experiments that neutrophils invade ANIT-treated livers in a CD18-independent fashion but utilize CD18 to induce hepatocellular cytotoxicity. The results emphasize that neutrophil-mediated amplification of ANIT-induced liver injury is directed toward hepatocytes rather than cholangiocytes. In fact, the data indicate that the majority of ANIT toxicity toward hepatocytes in vivo is neutrophil driven.

The pathogenic mycobacteria that cause tuberculosis (TB) and TB-like diseases in humans and animals elude sterilizing immunity by residing within an intracellular niche in host macrophages, where they are protected from microbicidal attack. Recent studies have emphasized microbial mechanisms for evasion of host defense; less is known about mycobactericidal mechanisms that remain intact during initial infection. To better understand macrophage mechanisms for restricting mycobacteria growth, we examined Mycobacterium marinum infection of Drosophila S2 cells. Among Ϸ1,000 host genes examined by RNAi depletion, the lysosomal enzyme ␤-hexosaminidase was identified as an important factor in the control of mycobacterial infection. The importance of ␤-hexosaminidase for restricting mycobacterial growth during mammalian infections was confirmed in macrophages from ␤-hexosaminidase knockout mice. ␤-Hexosaminidase was characterized as a peptidoglycan hydrolase that surprisingly exerts its mycobactericidal effect at the macrophage plasma membrane during mycobacteria-induced secretion of lysosomes. Thus, secretion of lysosomal enzymes is a mycobactericidal mechanism that may have a more general role in host defense.lysosome ͉ Mycobacterium marinum ͉ RNAi ͉ S2 ͉ bacterial pathogenesis