Fibrosis resolution in the mouse liver: Role of Mmp12 and potential role of calpain 1/2.

Although most work has focused on resolution of collagen ECM, fibrosis resolution involves changes to several ECM proteins. The purpose of the current study was twofold: 1) to examine the role of MMP12 and elastin; and 2) to investigate the changes in degraded proteins in plasma (i.e., the "degradome") in a preclinical model of fibrosis resolution. Fibrosis was induced by 4 weeks carbon tetrachloride (CCl4) exposure, and recovery was monitored for an additional 4 weeks. Some mice were treated with daily MMP12 inhibitor (MMP408) during the resolution phase. Liver injury and fibrosis was monitored by clinical chemistry, histology and gene expression. The release of degraded ECM peptides in the plasma was analyzed using by 1D-LC-MS/MS, coupled with PEAKS Studio (v10) peptide identification. Hepatic fibrosis and liver injury rapidly resolved in this mouse model. However, some collagen fibrils were still present 28d after cessation of CCl4. Despite this persistent collagen presence, expression of canonical markers of fibrosis were also normalized. The inhibition of MMP12 dramatically delayed fibrosis resolution under these conditions. LC-MS/MS analysis identified that several proteins were being degraded even at late stages of fibrosis resolution. Calpains 1/2 were identified as potential new proteases involved in fibrosis resolution. CONCLUSION. The results of this study indicate that remodeling of the liver during recovery from fibrosis is a complex and highly coordinated process that extends well beyond the degradation of the collagenous scar. These results also indicate that analysis of the plasma degradome may yield new insight into the mechanisms of fibrosis recovery, and by extension, new "theragnostic" targets. Lastly, a novel potential role for calpain activation in the degradation and turnover of proteins was identified.

Survey of the extracellular matrix architecture across the rat arterial tree.

OBJECTIVE: To understand arterial remodeling and the pathophysiology of arterial diseases, it is necessary to understand the baseline qualities and variations in arterial structure. Arteries could differ in wall thickness, laminar structure, and laminar fenestration depending on their position within the arterial tree. We endeavored to evaluate and compare the extracellular matrix structure of different arteries throughout the arterial tree, from the aorta to the adductor muscle arteriole, with a particular focus on the internal elastic lamina (IEL). METHODS: Arterial segments were harvested from male Sprague-Dawley rats and imaged using multiple modalities. En face scans by multiphoton microscopy were used to compare native-state adventitial collagen undulation and IEL fenestration. RESULTS: Collagen undulation was similar across most examined arteries but straighter in the skeletal muscle arterioles (P < .05). The elastic lamellae showed several differences. The IEL fenestrae were similar in average size among abdominal aorta and celiac, renal, common iliac, and common femoral arteries (range, 14-24 µm2), with wide within-vessel variance (square of the standard deviation, 462-1904 µm4). However, they tended to be smaller (9.08 µm2) and less variable (square of the standard deviation, 88.3 µm4) in the popliteal artery. Fenestrae were greater in number in the superior mesenteric artery (SMA; 6686/mm2; P < .05) and profunda femoris artery (PFA; 11,042/mm2; P < .05) compared with the other examined vessels, which ranged in surface density from 3143/mm2 to 4362/mm2. The SMA and PFA also showed greater total fenestration as a proportion of the IEL surface area (SMA, 15.04%; P < .05; PFA, 24.11%; P < .001) than the other examined arteries (range of means, 4.7%-9.4%). The arteriolar IEL was structurally distinct, comparable to a low-density wireframe. Other structural differences were also noted, including differences in the number of medial lamellae along the arterial tree. CONCLUSIONS: We found that vessels at different locations along the arterial tree differ in structure. The SMA, PFA, and intramuscular arterioles have fundamental differences in the extracellular matrix structure compared with other arteries. Location-specific features such as the medial lamellae number and elastic laminar structure might have relevance to physiology and confer vulnerabilities to the development of pathology.

Simultaneous Upregulation of Elastolytic and Elastogenic Factors Are Necessary for Regulated Collateral Diameter Expansion.

Background: During arteriogenesis, outward remodeling of the arterial wall expands luminal diameter to produce increased conductance in developing collaterals. We have previously shown that diameter expansion without loss of internal elastic lamina (IEL) integrity requires both degradation of elastic fibers and LOX-mediated repair. The aim of this study was to investigate the expression of genes involved in remodeling of the extracellular matrix (ECM) using a model of arteriogenesis. Methods: Sprague-Dawley rats underwent femoral artery ligation with distal arteriovenous fistula (FAL + AVF) placement. Profunda femoral arteries (PFA) were harvested for analysis at various time points. Serum desmosine, an amino acid found exclusively in elastin, was evaluated with enzyme-linked immunosorbent assay (ELISA) as a marker of tissue elastolysis. Tissue mRNA isolated from FAL + AVF exposed PFAs was compared to the contralateral sham-operated using qPCR. HCAECs were cultured under low shear stress (8 dyn·s/cm 2) for 24 h and then exposed to high shear stress (40 dyn·s/cm 2) for 2-6 h. Primers used included FBN-1, FBN-2, Timp-2, LOX-1, Trop-E, Cath-K, Cath-S, MMP-2, MMP-9, FBLN-4, and FBLN-5 and were normalized to GAPDH. mRNA fold changes were quantified using the 2-ΔΔCq method. Comparisons between time points were made with non-parametric ANOVA analysis with Bonferroni adjustment. Results: PFAs showed IEL reorganization during arteriogenesis. Serum desmosine levels are significantly elevated at 2 days and one week, with a return to baseline thereafter (p < 0.01). Expression of ECM structural proteins (FBN-1, FBN-2, FBLN-4, FBLN-5, Tropoelastin, TIMP-2, LOX-1) and elastolytic proteins (MMP-2, MMP-9, Cathepsin S, Cathepsin K) exhibited an early peak (p < 0.05) relative to sham PFAs. After two weeks, expression returned to baseline. HCAECs demonstrated upregulation of FBN-2, FBLN-5, LOX-1 and Trop-E at 4 h of high shear stress, as well as elastolytic protein MMP-2. Conclusions: Elastin degradation begins early in arteriogenesis and is mediated by local upregulation of elastolytic genes. Elastolysis appears to be simultaneously balanced by production of elastic fiber components which may facilitate stabilization of the IEL. Endothelial cells are central to initiation of arteriogenesis and begin ECM remodeling in response to altered shear stress.

Elastic Laminar Reorganization Occurs with Outward Diameter Expansion during Collateral Artery Growth and Requires Lysyl Oxidase for Stabilization.

When a large artery becomes occluded, hemodynamic changes stimulate remodeling of arterial networks to form collateral arteries in a process termed arteriogenesis. However, the structural changes necessary for collateral remodeling have not been defined. We hypothesize that deconstruction of the extracellular matrix is essential to remodel smaller arteries into effective collaterals. Using multiphoton microscopy, we analyzed collagen and elastin structure in maturing collateral arteries isolated from ischemic rat hindlimbs. Collateral arteries harvested at different timepoints showed progressive diameter expansion associated with striking rearrangement of internal elastic lamina (IEL) into a loose fibrous mesh, a pattern persisting at 8 weeks. Despite a 2.5-fold increase in luminal diameter, total elastin content remained unchanged in collaterals compared with control arteries. Among the collateral midzones, baseline elastic fiber content was low. Outward remodeling of these vessels with a 10-20 fold diameter increase was associated with fractures of the elastic fibers and evidence of increased wall tension, as demonstrated by the straightening of the adventitial collagen. Inhibition of lysyl oxidase (LOX) function with ß-aminopropionitrile resulted in severe fragmentation or complete loss of continuity of the IEL in developing collaterals. Collateral artery development is associated with permanent redistribution of existing elastic fibers to accommodate diameter growth. We found no evidence of new elastic fiber formation. Stabilization of the arterial wall during outward remodeling is necessary and dependent on LOX activity.