Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Abstract

Objective: We previously demonstrated that upregulation of intermediate-conductance Ca(2+)-activated K(+) channels (K(Ca)3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of K(Ca)3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis.

Methods and results: Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific K(Ca)3.1 blocker TRAM-34. Expression of K(Ca)3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. K(Ca)3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented K(Ca)3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level.

Conclusions: Blockade of K(Ca)3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis.

Figure 1. TRAM-34 prevents angioplasty-induced phenotypic modulation

Relative mRNA of K_Ca3.1 (a), REST (b), SMMHC (c), c-jun (d), c-fos (e), and myocardin (f) 2 hours (black bars) and 2 days (gray bars) postangioplasty in non-injured (Control), balloon injured (Injured), and TRAM-34–coated balloon injured (TRAM) coronary media. *P<0.05 vs corresponding control (n=4 to 5 per group).

Figure 2. K_Ca3.1 and SMMHC protein 2 days postangioplasty

Representative coronary sections isolated 2 days postangioplasty stained for K_Ca3.1 (a, HRP, pink) or SMMHC (b, DAB, brown). K_Ca3.1 staining was more intense (a, black arrowheads), whereas SMMHC staining was more diffuse (b white arrowheads) near the medial tear. Horizontal bar=100 µm.

Figure 3. K_Ca3.1 and SMMHC histology 2 hours and 2 days postangioplasty

Representative cross-sections (8 µm; 4 to 5 per group) of control, injured, and TRAM-34–coated balloon injured (TRAM) LCX and LAD 2 hours and 2 days postangioplasty exposed to antibodies against K_Ca3.1 (1:600, pink, top), Ki-67 (1:200, blue, top), and SMMHC (1:800, brown, bottom). Horizontal bar=100 µm.

Figure 4. REST histology 2 hours and 2 days postangioplasty

Representative cross-sections (8 µm; 4 to 5 per group) of control, injured, and TRAM-34–coated balloon injured (TRAM) LCX and LAD 2 hours and 2 days postangioplasty exposed to anti-REST (1:50,000, brown), and counterstained with hematoxylin (blue nuclei were considered REST negative). Horizontal bar=50 µm (25 µm in insets).

Figure 5. Histological analysis of K_Ca3.1, SMMHC, and REST

K_Ca3.1 (a), SMMHC (b), and REST (c) staining were quantified using Image Pro Plus. Injury-induced changes in K_Ca3.1, SMMHC, and REST staining were blocked by TRAM-34 (see Figure 3 and Figure 4 for representative images). *P<0.05 vs respective control and †P<0.05 vs injured and control (n=4 to 5 per group).

Figure 6. TRAM-34 prevents restenosis 14 and 28 days postangioplasty

Representative coronary arteries injured with a noncoated (a) or TRAM-34 coated balloon (b) 28 days postangioplasty and stained with Verhoff van Giesen stain (VVG). TRAM-34 reduced normalized intimal to medial thickness ratio (IMT/RI) at both 14 (38%) and 28 (22%) days postangioplasty (c). *P<0.05 vs injured segment taken from the same animal (paired t test; n=5). Horizontal bar=100 µm.