Publications

RATIONALE

Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.

OBJECTIVES

To determine the mechanisms by which autophagic proteins regulate tissue responses during PH.

METHODS

Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B(-/-)), or early growth response-1 (Egr-1(-/-)), were evaluated for vascular morphology and hemodynamics.

MEASUREMENTS AND MAIN RESULTS

Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.

CONCLUSIONS

The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B(-/-) and Egr-1(-/-) mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.

BACKGROUND

In the early highly active antiretroviral therapy (HAART) era, kidney dysfunction was strongly associated with death among HIV-infected individuals. We re-examined this association in the later HAART period to determine whether chronic kidney disease remains a predictor of death after HAART initiation.

METHODS

To evaluate the effect of kidney function at the time of HAART initiation on time to all-cause mortality, we evaluated 1415 HIV-infected women initiating HAART in the Women's Interagency HIV Study. Multivariable proportional hazards models with survival times calculated from HAART initiation to death were constructed; participants were censored at the time of the last available visit or December 31, 2006.

RESULTS

Chronic kidney disease (estimated glomerular filtration rate less than 60 mL/min/1.73 m) at HAART initiation was associated with higher mortality risk adjusting for age, race, hepatitis C serostatus, AIDS history, and CD4 cell count (hazard ratio 2.23, 95% confidence interval: 1.45-3.43). Adjustment for hypertension and diabetes history attenuated this association (hazard ratio = 1.89, confidence interval: 0.94-3.80). Lower kidney function at HAART initiation was weakly associated with increased mortality risk in women with prior AIDS (hazard ratio = 1.09, confidence interval: 1.00-1.19, per 20% decrease in estimated glomerular filtration rate).

CONCLUSIONS

Kidney function at HAART initiation remains an independent predictor of death in HIV-infected individuals, especially in those with a history of AIDS. Our study emphasizes the necessity of monitoring kidney function in this population. Additional studies are needed to determine mechanisms underlying the increased mortality risk associated with chronic kidney disease in HIV-infected persons.

Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has an unusual "Janus-faced" ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu(226) in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala(226) for Glu(226), suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild-type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr(4) over activation at Phe(8). Ala(226)Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala(226)Glu missense mutation appearing in primates 31-43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions.