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. 2010 Aug 13;299(5):L639–L651. doi: 10.1152/ajplung.00405.2009

Fig. 6.

Fig. 6.

Pulmonary vascular CD44 regulation of HMW-HA-induced protection from LPS-induced vascular hypermeability. A: immunohistochemical fluorescently stained images of control (untreated) mouse lung [bright-field (DIC) imaging (a) or treatment with anti-Factor VIII (von Willebrand factor) antibody (b), anti-caveolin-1 antibody (c), or FITC-conjugated anti-CD44 antibody (d) and secondary fluorescent antibody (Alexa Fluor 610 for vWF and 350 for caveolin-1; e)]. Magnification ×100. Arrows in e (an overlay of b, c, and d) indicate immunostaining of endothelial cells. Insets: negative controls for immunohistochemical analysis, which were obtained by the method described above, but without primary antibody. B: bronchoalveolar lavage (BAL) protein concentration from wild-type (C57BL/6J), CD44 knockout, or caveolin-1 knockout mice that were anesthetized and given saline (control) or LPS (2.5 mg/kg) intratracheally. After 4 h, mice were injected intravenously (internal jugular vein) with saline (control) or HMW-HA (1.5 mg/kg). Treated mice were allowed to recover for 24 h, BAL fluids were obtained, and protein concentrations were determined. *Significant (P < 0.05) difference between LPS and HMW-HA + LPS (n = 6 per condition). C: immunohistochemical analysis of murine lungs and kidneys either left untreated or injected intravenously (internal jugular vein) with angiotensin I-converting enzyme (ACE) antibody-conjugated liposomes (DOTAP/DOPE) containing siControl siRNA (10 mg/kg) or siSTABLE CD44 siRNA (10 mg/kg) for 5 days. Vascular CD44 expression is inhibited in lung, but not kidney. D: immunocytochemical analysis of cytospin material from BAL of mice injected intravenously (internal jugular vein) with ACE antibody-conjugated liposomes (DOTAP/DOPE) containing siControl siRNA (10 mg/kg) or siSTABLE CD44 siRNA (10 mg/kg) for 5 days. There is no difference in CD44 immunoreactivity. E: total BAL protein of B6129N2 mice injected intravenously (internal jugular vein) with ACE antibody-conjugated liposomes (DOTAP/DOPE) containing siControl siRNA (5 or 10 mg/kg) or siSTABLE CD44 siRNA (5 or 10 mg/kg) for 5 days (n = 6 per condition). *Statistically significant difference (P < 0.05) between siControl and siCD44. F: BAL protein concentration from C57BL/6J mice injected intravenously (internal jugular vein) with ACE antibody-conjugated liposomes (DOTAP/DOPE) containing scramble siRNA (10 mg/kg) or siSTABLE CD44 siRNA (10 mg/kg) for 5 days. Mice were then anesthetized and given saline (control) or LPS (2.5 mg/kg) intratracheally. After 4 h, mice were injected intravenously (internal jugular vein) with saline (control) or HMW-HA (1.5 mg/kg). Treated mice were allowed to recover for 24 h, BAL fluids were obtained, and protein concentrations were determined. *Significant (P < 0.05) difference between LPS and HMW-HA + LPS (n = 6 per condition).