Deletion of type VIII collagen reduces blood pressure, increases carotid artery functional distensibility and promotes elastin deposition

Figures

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  Fig. 1. Increased pressure-diameter curves, functional distensibility and phenylephrine-induced contractility in Col8−/−carotid arteries. A & D, Pressure myographs were obtained for carotid arteries maintained in either calcium or calcium-free MOPs buffer. Vessels were subjected to pressure increase from 0 to 160 mmHg, at 20 mmHg increments, and external diameter was recorded. B & E, Segmental distensibility and C & F, functional distensibility were calculated. G, Carotid arteries were maintained in calcium-containing MOPs buffer at 80 mmHg and subjected to increasing concentrations of PE from 10-8 M to 10-3 M. Changes in external diameter were recorded and percent constriction was calculated. Values represent the mean ± SEM. (A-F n = 8–11; G n = 5–8), *p < 0.05; **p < 0.01; ***p < 0.001.

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  Fig. 2. Differential VSMC and extracellular matrix protein expression in Col8+/+ and Col8−/−carotid arteries. A, Venn diagram illustrating the number of proteins shared by Col8+/+ and Col8−/− carotid arteries in ≥ 3 biological replicates per group. B, Heat map depicting the hierarchical clustering of proteins significantly different(p < 0.05). C, Volcano plot identifying differentially expressed VSMC and ECM proteins. D, elastin and E, collagen content were measured biochemically via desmosine and hydroxyproline assays respectively in Col8+/+ and Col8−/− carotid arteries. Values represent the mean ± SEM (A-C n = 4; for the proteomic studies each biological replicate contains the left and right carotid arteries pooled from 2 mice. We then used 4 biological replicates per genotype. D-E n = 9–12). *p < 0.05.

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  Fig. 3. Phenylephrine-induced phosphorylation of MYPT1 was enhanced in Col8−/−VSMCs, and smooth muscle cell marker expression was increased. A-B, serum starved Col8+/+ and Col8−/− VSMCs were stimulated with 10 µM PE for 10 min and immunoblotted for pMYPTI/MYPT1. C, Col8+/+ and Col8−/− VSMCs were immunoblotted for VMSC markers. Quantification for αSMA (D); SM22 (E); Calponin (F); SM-MHC (G) is depicted. All band intensities were normalized to β-actin. Values represent the mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001.

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  Fig. 4. Differential matrix composition in Col8+/+ and Col8−/−VSMCs in vitro. A-B, Expression levels for ECM genes related to elastic fiber proteins (Eln, Lox, Fbn1, Fbn2, Efemp2, Fbln5, Emilin1, Magp1), collagens (Col1a1, Col1a2, Col3a1, Col5a1, Col5a2, Col5a3), and fibronectin were determined by qRT-PCR. C, Decellularized matrices were labeled for either elastin, fibronectin or collagen-I. Scale bar = 100 µM. Mean intensity quantified for D, elastin (green); E, collagen-I (green); F, fibronectin (green). Values represent the mean ± SEM (n = 3), *p < 0.05; **p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 5. Exogenous rCol-8 reduced tropoelastin mRNA levels and elastic fiber deposition in Col8−/−VSMCs in vitro. A-B, Col8+/+ and Col8−/− VSMCs were cultured in the presence or absence of 2 µg/cm2 exogenous collagen VIII coated on the plates (-/+ VIII). Cells were labeled for elastin (green) and mean intensity quantified. Scale bar = 100 µM. C, mRNA was isolated from Col8+/+ and Col8−/− VSMCs cultured in the presence of exogenous collagen VIII (+VIII) and Eln expression levels were determined by qRT-PCR. Values represent the mean ± SEM (n = 3), ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 6. Working hypothesis model of Col-8 inhibiting elastin. The transcription, translation and formation of tropoelastin into elastic fibers is depicted by black arrows. Col-8 reduced tropoelastin mRNA levels and elastic fibers, as depicted by red inhibiting lines. Col-8 is hypothesized to perform these functions through α2β1 integrin signaling, which is outlined as a hashed red inhibiting line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)