A new role for the muscle repair protein dysferlin in endothelial cell adhesion and angiogenesis

Abstract

Objective: Ferlins are known to regulate plasma membrane repair in muscle cells and are linked to muscular dystrophy and cardiomyopathy. Recently, using proteomic analysis of caveolae/lipid rafts, we reported that endothelial cells (EC) express myoferlin and that it regulates membrane expression of vascular endothelial growth factor receptor 2 (VEGFR-2). The goal of this study was to document the presence of other ferlins in EC.

Methods and results: EC expressed another ferlin, dysferlin, and that in contrast to myoferlin, it did not regulate VEGFR-2 expression levels or downstream signaling (nitric oxide and Erk1/2 phosphorylation). Instead, loss of dysferlin in subconfluent EC resulted in deficient adhesion followed by growth arrest, an effect not observed in confluent EC. In vivo, dysferlin was also detected in intact and diseased blood vessels of rodent and human origin, and angiogenic challenge of dysferlin-null mice resulted in impaired angiogenic response compared with control mice. Mechanistically, loss of dysferlin in cultured EC caused polyubiquitination and proteasomal degradation of platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31), an adhesion molecule essential for angiogenesis. In addition, adenovirus-mediated gene transfer of PECAM-1 rescued the abnormal adhesion of EC caused by dysferlin gene silencing.

Conclusions: Our data describe a novel pathway for PECAM-1 regulation and broaden the functional scope of ferlins in angiogenesis and specialized ferlin-selective protein cargo trafficking in vascular settings.

Figure 1. Cultured EC express Dysferlin

A) Detection of a 1.5Kbp Dysferlin-specific amplicon by RT-PCR with primary BAEC or HUVEC mRNA as templates. Amplification was sensitive to RNAse treatment before RT. B) Northern blot analysis using BAEC and HUVEC total mRNA (left) showed positive Dysferlin mRNA expression (arrow; right). C) WB analysis of Dysferlin protein expression in HUVEC, BAEC and mouse skeletal muscle lysates using two antisera (Dysf Ab #1 and 2). Loading control was β-COP. D) Confocal optical section (xy plane) and sectional views (xz plane of nuclei and Golgi) showing GFP-Dysferlin expression in live BAEC. Color gradient is shown to illustrate GFP signal intensity levels. Scale bar 10 μm. E) Enrichment of Dysferlin in CEM/LR (light fractions) from HUVEC and BAEC lysates. Blotting against HSP90 and β-COP was performed to show lack of bulk and Golgi apparatus proteins in CEM/LR.

Figure 2. Dysferlin gene silencing causes loss of proliferation through impaired adhesion in sub-confluent but not confluent EC

A,B) Down-regulation of Dysferlin in near-confluent BAEC or HUVEC did not decrease VEGFR-2 or Tie-2 expression. Cells were treated with bovine or human-specific Dysferlin (Dysf1-2) or scrambled non-silencing siRNA (NS1-2). WB analysis against Dysferlin (top) or VEGFR-2 or Tie-2 following IP (IP) (bottom blots) was performed. C) Dysferlin gene silencing decreases BAEC proliferation. BAEC were seeded (approx. 5% confluency), transfected with siRNA sequences and starved in 0.1% FBS. Cells were then stimulated in 10% FBS or starved further and counted at 24, 48, or 72h. N = 8 in triplicate per condition. D) Loss of Dysferlin caused rapid defect in BAEC adhesion. Cells were treated as described in C and the adherent vs total cell ratio was determined by collecting unadhered cells and trypsinizing adhered cells for quantification. N = 6 per group in duplicate. E) Decreased angiogenesis following Dysferlin knock-down in vitro. Following siRNA treatment, confluent HUVEC were embedded in collagen gel with 10% FBS, and average length of tube-like structures per area (length mm/mm²) and proliferation (HUVEC/mm²) were quantified at 24 and 48 post-21 embedding. Typical data are shown, presented as mean +/- S.E.M. * P<0.001 compared to their respective non-silencing siRNA controls.

Figure 3. Dysferlin is expressed in blood vessels

A) Specificity of Dysferlin staining was confirmed by positive and negative Dysferlin detection in WT and Dysferlin-null mouse aorta paraffin sections, respectively. Scale bar 20 μm. B-D) Shown are 5 μm-thick adjacent sections of a human coronary artery with age-related hyperplasia (B; scale bar 100 μm), rat aorta (C; scale bar 25 μm) and mouse superficial femoral artery (D; scale bar 20 μm) stained with one of two Dysferlin antiserum (left image, brown color) or a non-immune IgG (right). Adventia (A), media (M) and neointima (NI). Counter-stain was performed with hematoxylin (blue). All immunostaining and image processing were performed in parallel, allowing direct comparison. Arrows indicate endothelial staining.

Figure 4. Dysferlin regulates neovascularisation

A) Genetic loss of Dysferlin caused abrogated angiogenic response to VEGF. Representative confocal optical sections (left) showing EC-positive structures (red channel) in ears of Dysferlin-null mice following adenoviral delivery of VEGF compared with age and sex-matched WT littermates. Control adenovirus encoded for or β-Gal. Blue channel represents DAPI (nucleus) staining. Scale bar 50 μm. Right, individual quantification of average total EC structures per ear section (n = 7 per group). *P < 0.05, **P < 0.01 compared with control virus, +P < 0.05 compared with WT. Black markers indicate ears shown on left panel. B) Adenoviral delivery of VEGF causes similar ear oedema in both WT and Dysferlin-null mice. Left, a section of the ears described in A was stained with H&E and midsection thickness was determined using a microscope. Black arrows describe thickness. Right, individual quantification of ear thickness. **P < 0.001 compared with control virus. Black markers indicate specimen depicted on left. C) Similar lung VEGFR-2 expression levels (240 and 210 kDa isoforms) in WT and Dysferlin-null mice, determined by IP and WB against VEGFR-2, or Dysferlin and HSP90 using the supernatant. Marker is 250 or 100 kDa.

Figure 5. Dysferlin gene silencing causes adhesion defect though PECAM-1 poly-ubiquitination and degradation

A) Decreased PECAM-1 expression following Dysferlin gene silencing in sub-confluent (left) but not confluent (right) BAEC. WB against Dysferlin, VE-Cadherin, PECAM-1 (marker 250 or 100 kDa) and HSP90 (loading control) was performed. B) Decreased PECAM-1 expression in sub-confluent HUVEC treated with human Dysferlin siRNA (24h; left). Dysferlin, PECAM-1 and β-COP expression (loading control) was analyzed by WB. Right, confocal immunofluorescence imaging (single optical sections) in 5 or 60% confluent HUVEC showing PECAM-1 (red) and nuclei (blue). Arrows indicate areas of high PECAM-1 staining and insets show magnified view of cell-cell junction. Scale bars 10 μm. C-D) PECAM-1 over-expression through adenovirus infection had no effect on adhesion of non-silencing siRNA-treated cells (solid markers) but rescued deficient adhesion by 59 and 68% in Dysferlin-silenced BAEC and HUVEC, respectively (approx 5% confluency time 0). * P<0.05, **P<0.01 compared with respective Adβ-Gal-treated control. E) Dysferlin silencing increased PECAM-1 poly-ubiquitination. Sub-confluent (25%) BAEC (left) and HUVEC (right) were transfected with a HA-ubiquitin plasmid and treated 24h later with control and Dysferlin siRNA sequences for 16h, proteins were collected and immuno-precipitated against PECAM-1 and blotted against HA. Marker (Mrk) is 100 (left) or 150 kDa (right). F) Inhibition of proteasome degradation with MG132 (6 h, 2×10^-6 M) partly rescued loss of PECAM-1 in sub-confluent cells (25%) treated with Dysferlin siRNA (24h). G) Dysferlin IP in HUVEC (25% confluent, top panel) resulted in PECAM-1 co-IP (bottom blot). IP with non-immune IgG control did not result in Dysferlin or PECAM recovery. Supernatant HSP90 was used as a loading control. H) Single confocal optical section showing GFP-Dysferlin (green) co-localization (yellow, arrows) with PECAM-1 (red) around the peri-nuclear regions and multiple cellular punctas (inset). Nucleus is shown in blue (DAPI). Scale bar 20 μm.

Figure 5. Dysferlin gene silencing causes adhesion defect though PECAM-1 poly-ubiquitination and degradation

A) Decreased PECAM-1 expression following Dysferlin gene silencing in sub-confluent (left) but not confluent (right) BAEC. WB against Dysferlin, VE-Cadherin, PECAM-1 (marker 250 or 100 kDa) and HSP90 (loading control) was performed. B) Decreased PECAM-1 expression in sub-confluent HUVEC treated with human Dysferlin siRNA (24h; left). Dysferlin, PECAM-1 and β-COP expression (loading control) was analyzed by WB. Right, confocal immunofluorescence imaging (single optical sections) in 5 or 60% confluent HUVEC showing PECAM-1 (red) and nuclei (blue). Arrows indicate areas of high PECAM-1 staining and insets show magnified view of cell-cell junction. Scale bars 10 μm. C-D) PECAM-1 over-expression through adenovirus infection had no effect on adhesion of non-silencing siRNA-treated cells (solid markers) but rescued deficient adhesion by 59 and 68% in Dysferlin-silenced BAEC and HUVEC, respectively (approx 5% confluency time 0). * P<0.05, **P<0.01 compared with respective Adβ-Gal-treated control. E) Dysferlin silencing increased PECAM-1 poly-ubiquitination. Sub-confluent (25%) BAEC (left) and HUVEC (right) were transfected with a HA-ubiquitin plasmid and treated 24h later with control and Dysferlin siRNA sequences for 16h, proteins were collected and immuno-precipitated against PECAM-1 and blotted against HA. Marker (Mrk) is 100 (left) or 150 kDa (right). F) Inhibition of proteasome degradation with MG132 (6 h, 2×10^-6 M) partly rescued loss of PECAM-1 in sub-confluent cells (25%) treated with Dysferlin siRNA (24h). G) Dysferlin IP in HUVEC (25% confluent, top panel) resulted in PECAM-1 co-IP (bottom blot). IP with non-immune IgG control did not result in Dysferlin or PECAM recovery. Supernatant HSP90 was used as a loading control. H) Single confocal optical section showing GFP-Dysferlin (green) co-localization (yellow, arrows) with PECAM-1 (red) around the peri-nuclear regions and multiple cellular punctas (inset). Nucleus is shown in blue (DAPI). Scale bar 20 μm.