Extracellular calpastatin protects against hypoxia-induced pulmonary hypertension (PH) in mice and is elevated in human PH - 04/04/15

Ubiquitous calpains 1 and 2 are calcium-activated neutral cysteine proteases that were shown to be involved in the progression of pulmonary hypertension (PH). Whether calpains contribute to the pulmonary vascular remodeling process through intracellular- or extracellular-mediated effects remains unclear. Here, we investigated whether overexpression of calpastatin, the specific endogenous calpain inhibitor, affected PH progression in mice through intracellular and/or extracellular mechanisms.

To specify the role for intracellular versus extracellular calpains during PH progression, we used transgenic mice ubiquitously overexpressing intracellular calpastatin under the control of the CMV promoter (CMV-cast) and transgenic mice with hepatic specific/inflammation-inducible expression of secreted calpastatin under the control of the human CRP promoter (CRP-cast).

Hypoxia exposure of wild-type (WT) mice led to marked increases in lung calpain 1 and 2 protein levels and activity, both intracellularly (as assessed by the spectrin breakdown product) and extracellularly (as assessed by fibronectin breakdown product), which peaked on day 8 and remained elevated until day 18. Calpastatin protein increased from day 1 to day 3 and returned to basal levels by day 18. Immunofluorescence studies revealed prominent calpain 1 and 2 staining in pulmonary-artery smooth muscle cells (PA-SMCs) of remodeled vessels. Both CRP-cast and CMV-cast mice showed decreased PH severity compared to WT mice, with similar decreases in right ventricular systolic pressure (RVSP), RV hypertrophy, and distal pulmonary-artery muscularization. Extracellular calpain activity was decreased only in CRP-cast mice. A selective calpain inhibitor, PD150606, protected WT mice against hypoxic PH but did not produce additional effects in CMV-cast or CRP-cast mice. PD150606 also partially reversed PH in SM22-5HTT+ mice, which spontaneously develop PH. The growth rate of PA-SMCs from CMV-cast mice was decreased compared to PA-SMCs from CRP-cast and WT mice. Treatment of PA-SMCs with PD150606 or calpastatin decreased PA-SMC proliferation and abolished the difference in cell growth rate across the three groups of mice. A role for extracellular calpastatin was shown in patients with idiopathic pulmonary arterial hypertension, who exhibited increased calpastatin pulmonary vessel immunostaining and increased circulating calpastatin levels compared to healthy controls.

These data indicate that both intracellular and extracellular calpains play an important role in the pathogenesis of PH and that calpastatin protects against PH by counteracting these effects.