Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling - PubMed (original) (raw)

Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling

Nesrine El-Bizri et al. Circ Res. 2008.

Abstract

Vascular expression of bone morphogenetic type IA receptor (Bmpr1a) is reduced in lungs of patients with pulmonary arterial hypertension, but the significance of this observation is poorly understood. To elucidate the role of Bmpr1a in the vascular pathology of pulmonary arterial hypertension and associated right ventricular (RV) dysfunction, we deleted Bmpr1a in vascular smooth muscle cells and in cardiac myocytes in mice using the SM22alpha;TRE-Cre/LoxP;R26R system. The LacZ distribution reflected patchy deletion of Bmpr1a in the lung vessels, aorta, and heart of SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutants. This reduction in BMPR-IA expression was confirmed by Western immunoblot and immunohistochemistry in the flox/flox group. This did not affect pulmonary vasoreactivity to acute hypoxia (10% O2) or the increase in RV systolic pressure and RV hypertrophy following 3 weeks in chronic hypoxia. However, both SM22alpha;TRE-Cre;R26R;Bmpr1a(flox/+) and flox/flox mutant mice had fewer muscularized distal pulmonary arteries and attenuated loss of peripheral pulmonary arteries compared with age-matched control littermates in hypoxia. When Bmpr1a expression was reduced by short interference RNA in cultured pulmonary arterial smooth muscle cells, serum-induced proliferation was attenuated explaining decreased hypoxia-mediated muscularization of distal vessels. When Bmpr1a was reduced in cultured microvascular pericytes by short interference RNA, resistance to apoptosis was observed and this could account for protection against hypoxia-mediated vessel loss. The similar elevation in RV systolic pressure and RV hypertrophy, despite the attenuated remodeling with chronic hypoxia in the flox/flox mutants versus controls, was not a function of elevated left ventricular end diastolic pressure but was associated with increased periadventitial deposition of elastin and collagen, potentially influencing vascular stiffness.

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Figures

Figure 1. Whole-mount LacZ staining of SM22α;TRE-Cre;R26R; Bmpr1aflox/+ and flox/flox KO embryos and adult mice and their controls

Patchy LacZ staining shown in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ KO E11.5 embryonic tissues (B, D, E, F) as indicated by red arrows as compared to control littermates not expressing R26R with absence of staining (A, C). RA: right atrium. Patchy staining in large (G and H) and distal PAs (I) in sections of whole-mount LacZ stained lungs of an adult control mouse and in the main PA (J) and heart (K, L) of whole-mount LacZ stained lungs and heart of SM22α;TRE-Cre;R26R;Bmpr1aflox/flox adult KO mouse. H is an enlargement of the outlined area in G and * identifies the lung airway. L represents a section of the heart at the level of the line indicated in K. Whole-mount immunofluorescence revealed a global reduction in BMPR-IA expression (red) in the aorta of SM22α;TRE-Cre; Bmpr1aflox/flox adult KO mouse (N) as compared to WT adult mouse (N). Note the clustered BMPR-IA expression in both WT and KO aortae. No staining was seen in the negative control (O). The blue color in M and N represents DAPI nuclear staining. Bars (I and L)= 50 μm, (G and H)= 100 μm. P. On the left is a western immunoblot of whole heart tissue showing BMPR-IA relative to S6 protein expression in two representative WT and flox/flox adult mice. Densitometric quantification of values from three WT and four flox/flox adult mice is depicted graphically on the right. Bars represent mean±SEM. *P<0.05.

Figure 2. Comparable pulmonary vasoreactivity in SM22α;TRE-Cre;R26R; Bmpr1aflox/+ and flox/flox KO adult mice and their controls under acute hypoxia

RVSP was measured in anesthetized SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice and age-matched controls under baseline 40% O2, in response to hypoxia (10% O2) for 15 min, and upon return to 40% O2 for 10 min. Bars represent mean±SEM (n=6-8). **P<0.01 and ***P<0.001 compared to normoxia; †P<0.05 and †††P<0.001, recovery compared to hypoxia.

Figure 3. Right ventricular systolic pressure (RVSP) and cardiac function in adult mice following 3 weeks of normoxia or chronic hypoxia

RVSP (A), max and min dP/dt (B), and fractional shortening (FS; C) were measured in anesthetized SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice and age-matched control littermates in normoxia and following three weeks of chronic hypoxia (10% O2). Bars represent mean±SEM (A and B, n=5-11; C, n=3-5). *P<0.05, **P<0.01, and ***P<0.001 between hypoxia and normoxia for each genotype; † P<0.05 and †† P<0.01 between the mutants and WT in chronic hypoxia.

Figure 4. Comparable right ventricular hypertrophy (RVH) but depressed vascular remodeling of SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox adult KO mice compared to controls following chronic hypoxia

RVH (A), % of partially and fully muscularized per total number of distal arteries (B), and number of distal arteries per mm2 (C) in SM22α;TRE-Cre;R26R;Bmpr1aflox/+ and flox/flox KO mice in normoxia and following three weeks of chronic hypoxia (10% O2). Panels in B show EVG-stained paraffin sections of barium-injected lungs where arteries were identified by barium filling (grey color). Note the thick vessel walls in controls compared to the thin vessel walls seen in the KO (red arrows). Bars represent mean±SEM (A, n=5-13; B, n=5-9; C, n=5-9). *P<0.05, **P<0.01, and ***P<0.001 between hypoxia and normoxia for each genotype; †P<0.05 between the mutants and WT in chronic hypoxia.

Figure 5. Changes in pulmonary vessel wall structure in chronic hypoxia

Quantitative measurements of wall thickness (A), external diameter (B), and internal lumen (C) in pre-acinar large PAs (≤200 μm) in lung sections of SM22α;TRE-Cre;R26R;Bmpr1aflox/flox KO mice and age-matched controls following 3 weeks of hypoxia versus normoxia. Bars represent mean±SEM (n=5-9). *P<0.05. D-K Lung sections of flox/flox KO and control mice in normoxia (upper panels) and chronic hypoxia (lower panels). D-G Hart’s elastin staining of sections of whole-mount LacZ stained lungs. H-K Masson’s trichrome (collagen) staining. Note the ectopic elastin deposition in brown color neighboring areas of Cre activity (green color, G) as well as compact collagen deposition in proximal large vessels of SM22α;TRE-Cre;R26R;Bmpr1a flox/flox KO lungs under chronic hypoxia (K). Panels D-G and H-K were acquired with the same magnification as D and H respectively; Bars in D and H are 100 μm.

Figure 6. Knock-down of Bmpr1a attenuates proliferation and survival of HPASMC and promotes survival of HBVP

A. HPASMC were transfected with siControl (SiCtrl) and siBmpr1a and the relative message transcript levels of Bmpr1a normalized to β2M. Bars represent mean±SEM (n=3 experiments run in quadruplicates). **P<0.01. B-D. Bmpr1a in HPASMC or HBVP was knocked-down using siBmpr1a for 48 hr. B. Proliferation of SiControl and SiBmpr1a HPASMC in presence of 10% FBS for 72 hr by MTT assay. Bars represent mean±SEM of fold increase in OD570nm readings relative to baseline SiControl cells (n=14-16). ***P<0.001. C. Apoptosis of SiControl and SiBmpr1a HPASMC in response to 7.7 nmol/L (200 ng/ml) BMP2 by TUNEL assay. D. Apoptosis of SiControl and SiBmpr1a HBVP in response to a total of 72 hr of serum deprivation by TUNEL assay. Bars in C and D represent mean±SEM of percentages of TUNEL positive cells over total number of cells labeled with nuclei-staining DAPI (stained in red and blue in their corresponding microscopic views, respectively) (n=4 for both). *P<0.05; **P<0.01.

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Smooth muscle protein 22alpha-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling - PubMed (original) (raw)