Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension - PubMed (original) (raw)

Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

M Sean McMurtry et al. J Clin Invest. 2005 Jun.

Abstract

Pulmonary arterial hypertension (PAH) is characterized by genetic and acquired abnormalities that suppress apoptosis and enhance cell proliferation in the vascular wall, including downregulation of the bone morphogenetic protein axis and voltage-gated K+ (Kv) channels. Survivin is an "inhibitor of apoptosis" protein, previously thought to be expressed primarily in cancer cells. We found that survivin was expressed in the pulmonary arteries (PAs) of 6 patients with PAH and rats with monocrotaline-induced PAH, but not in the PAs of 3 patients and rats without PAH. Gene therapy with inhalation of an adenovirus carrying a phosphorylation-deficient survivin mutant with dominant-negative properties reversed established monocrotaline-induced PAH and prolonged survival by 25%. The survivin mutant lowered pulmonary vascular resistance, RV hypertrophy, and PA medial hypertrophy. Both in vitro and in vivo, inhibition of survivin induced PA smooth muscle cell apoptosis, decreased proliferation, depolarized mitochondria, caused efflux of cytochrome c in the cytoplasm and translocation of apoptosis-inducing factor into the nucleus, and increased Kv channel current; the opposite effects were observed with gene transfer of WT survivin, both in vivo and in vitro. Inhibition of the inappropriate expression of survivin that accompanies human and experimental PAH is a novel therapeutic strategy that acts by inducing vascular mitochondria-dependent apoptosis.

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Figures

Figure 1

Figure 1

Survivin is expressed in the PAs of patients with PAH, but not the PAs of patients with secondary pulmonary hypertension or normal PAs. (A) Top: Immunofluorescence confocal microscopy shows survivin expression colocalizing with smooth muscle actin in the media of small and medium-sized PAs from 5 patients with PAH. In some cases (patients 3–5) survivin is expressed in cells both outside and inside the elastic lamina (evident by autofluorescence in patient 3). Bottom: In 2 small PAs shown from a PAH patient, survivin is also expressed; it colocalizes with vWF only in the bottom one, which appears to be more remodeled and have an almost obliterated lumen. (B) Survivin is not expressed in the PAs of 3 patients without PAH (patients 8 and 9; patient 10 not shown). Despite the significant medial hypertrophy shown in patient 7, due to thromboembolic pulmonary hypertension, there is a compete lack of survivin expression, suggesting that it is not a nonspecific feature of the remodeling process. Lack of nonspecific staining by the secondary antibodies in patient 4 is shown in the bottom; this was the case in all the patients shown here. SMA, smooth muscle actin; S, survivin; L, lumen.

Figure 2

Figure 2

Survivin is expressed in the PAs of rats with MCT-PAH, and its expression parallels the rise in PA pressure. (A) Survivin is expressed in the media of resistance PAs from rats with MCT-PAH but not in PAs from control rats. Note the heavy expression of survivin in severe MCT-PAH (21 days after MCT), compared with mild MCT-PAH (12 days after MCT). In the latter, note that a smooth muscle actin–positive PASMC is heavily expressing survivin in the media of a small PH that shows early medial hypertrophy, whereas no survivin is expressed in another PA from the same field that does not show evidence of medial hypertrophy. Magnification in S+SMA+DAPI, ×75. (B) Survivin expression, measured by quantitative RT-PCR and immunoblots, increases 10 days after MCT injection, prior to the increase of PA pressure as measured by in vivo telemetry (mean PA pressure [PAP] shown) and by echocardiography (PAAT). Kv1.5 expression is decreasing in parallel with survivin, before the rise in pressure, while bcl-2 expression is unchanged.

Figure 3

Figure 3

Efficient adenoviral delivery of phosphorylation-deficient survivin (Ad-GFP-S-M) to PASMCs in vitro reduces proliferation and increases apoptosis. (A) Primary culture of rat PASMCs stains positive for smooth muscle actin but not vWF, indicating no contamination with endothelial cells. (B) Infection of PASMCs with adenoviruses encoding GFP and either WT survivin (Ad-GFP-S) or survivin mutant (Ad-GFP-S-M) was highly efficient, as evidenced by GFP reporter (green fluorescence in the left panels and differential interference contrast [DIC] in the right panels). Note the reduced cellularity of the plate infected with Ad-GFP-S-M, compared with Ad-GFP-S. (C) Cells infected with Ad-GFP-S-M (grown in 10% FBS) show increased TUNEL-positive nuclei and reduced PCNA-positive nuclei, suggesting that they undergo apoptosis and not proliferation. In contrast, cells infected with Ad-GFP-S (grown in 0.1% FBS) show no apoptosis and increased rates of PCNA expression. (D) Mean data for TUNEL- and PCNA-positive nuclei, 48 hours after infection with Ad-GFP-S-M versus Ad-GFP-S; 5 fields studied in each plate, 20 plates per group. *P < 0.01.

Figure 4

Figure 4

Ad-GFP-S-M infection induces PASMC mitochondria-dependent apoptosis. (A) PASMCs effectively infected with Ad-GFP-S (green) show slight hyperpolarization of mitochondrial membrane potential (increased TMRM fluorescence) compared with noninfected cells. In contrast, mitochondria of Ad-GFP-S-M–infected cells (but not neighboring noninfected cells) are less red, indicating depolarized mitochondria. Mean data are shown on the right (arbitrary fluorescence units [FU], means from 15 plates per group; *P < 0.01). Immunoblots show that, in contrast to expression of WT survivin, expression of survivin mutant in PASMCs grown in 10% FBS induces activation of caspase-9 and caspase-3. (B) PASMCs infected with Ad-GFP-S demonstrate sequestered cytochrome c within mitochondria, as shown by the punctate pattern of staining. In contrast, mitochondria of Ad-GFP-S-M–infected cells show cytochrome c–positive staining diffusely throughout the cell, indicating leakage of cytochrome c from mitochondria into the cytoplasm. Magnification: left and middle panels, ×75; right panels, ×125. (C) In contrast to infection with Ad-GFP-S, infection with Ad-GFP-S-M induces translocation of the mitochondria-based apoptosis-inducing factor (AIF) in the nucleus, where it initiates caspase-independent apoptosis. Magnification, ×100.

Figure 5

Figure 5

Selective expression of survivin mutant in resistance PAs causes an increase in PASMC outward K+ current. (A) In FBS-rich conditions (10% FBS in the medium, a condition known to increase endogenous survivin), infection with Ad-GFP-S-M causes augmentation of K+ currents and decreased capacitance (Cm, a measure of cell size), consistent with apoptosis; the opposite is seen with Ad-GFP-S infection. In contrast, in serum-deprived conditions (0.1% FBS), infection with Ad-GFP-S causes a decrease in K+ currents, consistent with apoptosis resistance. Since in these conditions endogenous survivin is absent, infection with Ad-GFP-S-M has no effect on K+ current. Cells carrying the transgenes were selected by the green fluorescence. Mean data for current density over voltage are shown on the right (n = 6 cells per group; *P < 0.01 vs. control). (B) Both GFP immunofluorescence microscopy and quantitative RT-PCR of laser-capture-microdissected resistance PAs demonstrate efficient delivery of the transgenes, particularly to the very small (less than 50 μm) resistance PAs (arrows). (C) In our inhaled gene therapy approach, the expression of the transgenes is restricted to the lungs, as shown by the expression of GFP, measured by quantitative RT-PCR. The expression of endogenous survivin in nontreated rats is minimal in all organs studied, except the spleen. Our WT-survivin primer also detects the survivin mutant, as shown by the increased lung signal in the treated rats. Data from 5 rats per group are shown.

Figure 6

Figure 6

Gene therapy of rat MCT-PAH with Ad-GFP-S-M improves hemodynamics, reduces remodeling of the resistance PAs, and prolongs survival. (A) Representative high-fidelity PA pressure tracings and mean data show that Ad-GFP-S-M, but not Ad-GFP, therapy reduces PA pressure and PVRi, without altering systemic hemodynamics. SVRi, systemic vascular resistance index. (B and C) Ad-GFP-S-M, but not Ad-GFP, reduces RV thickness measured in parasternal short axis (see also Table 2) and preserves the normal round shape of the LV. Similarly, Ad-GFP-S-M reduces PAAT. Resistance PA remodeling, as measured by percent medial thickness, is reduced by treatment with Ad-GFP-S-M. RVOT, RV outflow tract; AV, aortic valve; PV, pulmonary valve. Magnification in C, ×40. (D) Targeting survivin with inhaled gene therapy in MCT-PAH significantly prolongs survival within the study period. *P < 0.05 vs. MCT; †P < 0.05 vs. Ad-GFP.

Figure 7

Figure 7

Ad-GFP-S-M augments apoptosis and Kv current and reduces proliferation within resistance PAs in vivo. (A) The number of TUNEL-positive nuclei (arrows) is increased by Ad-GFP-S-M treatment, while the number of PCNA-positive nuclei is reduced, compared with those in the Ad-GFP–treated and nontreated MCT-PAH rats. *P < 0.05 vs. MCT; †P < 0.05 vs. Ad-GFP. (B) Reduced BrdU staining (green) in resistance PAs of rats treated with Ad-GFP-S-M, compared with Ad-GFP-S (a representative image from 5 rats per group is shown). (C) Freshly isolated PASMCs from rats treated with Ad-GFP-S-M have increased K+ currents, in agreement with our in vitro data (Figure 5A). The sensitivity to 4-aminopyridine (4-AP; 5 mM) and current morphology suggest that the induced current is voltage-gated (Kv). *P < 0.05 vs. Ad-GFP.

Figure 8

Figure 8

Exogenous WT survivin, delivered by Ad-GFP-S in normal rats, induces PAH and medial hypertrophy, within 2 weeks from infection. Magnification, ×10.

Figure 9

Figure 9

Schematic linking mitochondria, survivin, and Kv channels as potential therapeutic targets for the regression of pulmonary vascular remodeling. Cyt c, cytochrome c.

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References

    1. Archer S, Rich S. Primary pulmonary hypertension: a vascular biology and translational research “work in progress”. Circulation. 2000;102:2781–2791. - PubMed
    1. Rubin LJ. Therapy of pulmonary hypertension: the evolution from vasodilators to antiproliferative agents. Am. J. Respir. Crit. Care Med. 2002;166:1308–1309. - PubMed
    1. Thomson JR, et al. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family. J. Med. Genet. 2000;37:741–745. - PMC - PubMed
    1. Lane KB, et al. Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat. Genet. 2000;26:81–84. - PubMed
    1. Zhang S, et al. Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003;285:L740–L754. - PubMed

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