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.

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.

Unbiased, High-Throughput Electron Microscopy Analysis of Experience-Dependent Synaptic Changes in the Neocortex.

Neocortical circuits can be altered by sensory and motor experience, with experimental evidence supporting both anatomical and electrophysiological changes in synaptic properties. Previous studies have focused on changes in specific neurons or pathways-for example, the thalamocortical circuitry, layer 4-3 (L4-L3) synapses, or in the apical dendrites of L5 neurons- but a broad-scale analysis of experience-induced changes across the cortical column has been lacking. Without this comprehensive approach, a full understanding of how cortical circuits adapt during learning or altered sensory input will be impossible. Here we adapt an electron microscopy technique that selectively labels synapses, in combination with a machine-learning algorithm for semiautomated synapse detection, to perform an unbiased analysis of developmental and experience-dependent changes in synaptic properties across an entire cortical column in mice. Synapse density and length were compared across development and during whisker-evoked plasticity. Between postnatal days 14 and 18, synapse density significantly increases most in superficial layers, and synapse length increases in L3 and L5B. Removal of all but a single whisker row for 24 h led to an apparent increase in synapse density in L2 and a decrease in L6, and a significant increase in length in L3. Targeted electrophysiological analysis of changes in miniature EPSC and IPSC properties in L2 pyramidal neurons showed that mEPSC frequency nearly doubled in the whisker-spared column, a difference that was highly significant. Together, this analysis shows that data-intensive analysis of column-wide changes in synapse properties can generate specific and testable hypotheses about experience-dependent changes in cortical organization. SIGNIFICANCE STATEMENT: Development and sensory experience can change synapse properties in the neocortex. Here we use a semiautomated analysis of electron microscopy images for an unbiased, column-wide analysis of synapse changes. This analysis reveals new loci for synaptic change that can be verified by targeted electrophysiological investigation. This method can be used as a platform for generating new hypotheses about synaptic changes across different brain areas and experimental conditions.