Multiple subregions within the caveolin-1 scaffolding domain inhibit fibrosis, microvascular leakage, and monocyte migration

Abstract

The caveolin-1 scaffolding domain (CSD, amino acids 82-101 of caveolin-1) has been shown to suppress bleomycin-induced lung and skin fibrosis and angiotensin II (AngII)-induced myocardial fibrosis. To identify active subregions within CSD, we split its sequence into three slightly overlapping 8-amino acid subregions (82-89, 88-95, and 94-101). Interestingly, all three peptides showed activity. In bleomycin-treated mice, all three subregions suppressed the pathological effects on lung and skin tissue morphology. In addition, while bone marrow monocytes isolated from bleomycin-treated mice showed greatly enhanced migration in vitro toward CXCL12, treatment in vivo with CSD and its subregions almost completely suppressed this enhanced migration. In AngII-induced heart failure, both 82-89 and 88-95 significantly suppressed fibrosis (both Col I and HSP47 levels), microvascular leakage, and heart weight/ body weight ratio (HW/BW) while improving ventricular function. In contrast, while 94-101 suppressed the increase in Col I, it did not improve the other parameters. The idea that all three subregions can be active depending on the assay was further supported by experiments studying the in vitro migration of human monocytes in which all three subregions were extremely active. These studies are very novel in that it has been suggested that there is only one active region within CSD that is centered on amino acids 90-92. In contrast, we demonstrate here the presence of other active regions within CSD.

Fig 1. Inhibition of bleomycin-induced lung fibrosis and monocyte migration by subregions of CSD.

A disease state similar to human SSc was induced by systemic treatment of mice with bleomycin [47]. Following removal of the bleomycin-containing pumps on day 8, treatment was initiated using pumps implanted subcutaneously loaded with 8 μmol/kg of the indicated peptides that release their contents for 2 weeks. (A) Masson’s Trichrome-stained lung tissue sections demonstrate the massive fibrosis caused by bleomycin and its reversal by 82–89 both in the edge and interior of a lung lobe. (B) Tissue Morphology in Masson’s Trichrome-stained tissue sections was quantified as described [16, 49]. (n = 6 per group) (C) Monocyte migration in vitro was quantified as described [21] using BM monocytes isolated from mice treated in vivo both with bleomycin and with peptides as indicated. (n = 4 per group) *** p<0.001 vs Sham control; ^^^ p<0.001 vs Bleo control.

Fig 2. Dose-dependent effect of CSD subregions on human monocyte migration.

Healthy human monocytes were treated with the indicated versions and concentrations of CSD. 100 ng/ml SDF-1 was the chemoattractant. Migrating cells were counted in 6 high power fields per filter. Each experiment included control (unstimulated) monocytes and the same monocytes treated with TGFβ, which enhanced their migration ~3-fold. Inhibition of migration is presented on a linear scale of 0.0 (no effect) to 1.0 (reduction to level observed in absence of TGFβ). Results are summarized from 11 independent experiments.

Fig 3. Inhibition of bleomycin-induced dermal fibrosis and thinning of the transdermal fat layer (lipoatrophy) by subregions of CSD.

The same mice used in Fig 1 were used here. After sacrifice, the thickness of the dermis and intradermal fat layer in tissue sections were measured (six mice per condition). (A) Masson’s Trichrome-stained skin tissue sections demonstrate the dermal fibrosis and lipoatrophy caused by bleomycin and its reversal by 82–89. (B) Quantification of dermal fibrosis. (C) Quantification of lipoatrophy. ***p<0.001 vs Saline control; ^^^p<0.001, ^^p<0.01, ^p<0.05 vs Bleo control.

Fig 4. CSD and two subregions suppress Ang II-induced cardiac hypertrophy and pathological changes in ventricular function.

Mice were infused for 2 weeks with Ang II (2.1 mg/kg/day) to induce HF or with vehicle. Concomitantly, CSD, scrambled CSD, or subregions were injected daily (i.p., 0.8 μmol/kg). Cardiac hypertrophy was evaluated in terms of the HW/BW ratio. Cardiac function was evaluated by echocardiography for IVRT, EF, FS, SV, and CO. Significant changes are shown as ***p<0.001 vs Sham control. Suppression of AngII-induced changes is shown as ^p<0.05, ^^p<0.01, and ^^^p<0.001 vs AngII control. Additionally, representative images of echocardiography data are provided in the Supplementary figures.

Fig 5. Inhibition of AngII-Induced fibrosis and microvascular leakage by CSD subregions.

(A) HF induction and peptide treatment were as in Fig 4. Fibrosis was evaluated in LV tissue extracts by Western blot of Col I levels in the RIPA-insoluble fraction and HSP47 levels in the RIPA-soluble fraction. Microvascular leakage was evaluated in terms of IgG Heavy chain levels in the RIPA-soluble fraction. Actin in each fraction served as the loading control. n = 3 in this experiment. Similar results were obtained in a second experiment. (B) Quantification of Western blot results. Significant changes are shown as *p < 0.05 and **p<0.01 vs Sham control. Suppression of Ang II-induced changes by subregions are shown as ^p<0.05, ^^p< 0.01, and ^^^p<0.001 vs Ang II control.