Background-Interstitial collagen plays a crucial structural role in arteries. Matrix metalloproteinases (MMPs), including MMP-13/collagenase-3, likely contribute to collagen catabolism in atherosclerotic plaques. Methods and Results-To test the hypothesis that a specific MMP-collagenase influences the development and structure of atherosclerotic plaques, this study used atherosclerosis-susceptible apolipoprotein E-deficient mice that lack MMP-13/collagenase-3 (Mmp-13 Ϫ/Ϫ /apoE Ϫ/Ϫ ) or express wild-type MMP-13/collagenase-3 (Mmp-13 ϩ/ϩ /apoE Ϫ/Ϫ ). Both groups consumed an atherogenic diet for 5 (nϭ8) or 10 weeks (nϭ9). Histological analyses of the aortic root of both groups revealed similar plaque size and accumulation of smooth muscle cells (a collagen-producing cell type) and macrophages (a major source of plaque collagenases) after 5 and 10 weeks of atherogenic diet. By 10 weeks, the plaques of Mmp-13

Background-We hypothesized that collagenolytic activity produced by activated macrophages contributes to collagen loss and the subsequent instability of atheromatous lesions, a common trigger of acute coronary syndromes. However, no direct in vivo evidence links collagenases with the regulation of collagen content in atherosclerotic plaques. Methods and Results-To test the hypothesis that collagenases influence the structure of atheromata, we examined collagen accumulation in atherosclerotic lesions of apolipoprotein E-deficient mice (apoE Ϫ/Ϫ ) that express collagenase-resistant collagen-I (Col R/R /apoE Ϫ/Ϫ , nϭ12) or wild-type collagen-expressing mice (Col ϩ/ϩ /apoE Ϫ/Ϫ , nϭ12). Aortic atheromata of both groups had similar sizes and numbers of macrophages, a major source of collagenases. However, aortic intimas from Col R/R /apoE Ϫ/Ϫ mice contained fewer smooth muscle cells, a source of collagen, probably because of decreased migration or proliferation or increased cell death. Despite reduced numbers of smooth muscle cells, atheromata of Col R/R /apoE Ϫ/Ϫ mice contained significantly more intimal collagen than did those of Col ϩ/ϩ /apoE Ϫ/Ϫ mice. Conclusion-These results establish that collagenase action regulates plaque collagen turnover and smooth muscle cell accumulation.

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Clinical evidence links increased aortic collagen content and stiffness to abdominal aortic aneurysm (AAA) formation. However, the possibility that excess collagen contributes to AAA formation remains untested. We investigated the hypothesis that augmented collagen promotes AAA formation, and employed apoE-null mice expressing collagenaseresistant mutant collagen (Col R/R /apoE À/À ), heterozygote (Col R/ þ /apoE À/À ), or wild-type collagen (Col þ / þ /apoE À/À ) infused with angiotensin II to induce AAA. As expected, the aortas of Col R/R /apoE À/À mice contained more interstitial collagen than those from the other groups. Angiotensin II treatment elicited more AAA formation in Col R/R /apoE À/À mice than Col R/ þ /apoE À/À or Col þ / þ /apoE À/À mice. Aortic circumferences correlated positively with collagen content, determined by picrosirius red and Masson trichrome staining. Mechanical testing of aortas of Col R/R /apoE À/À mice showed increased stiffness and susceptibility to mechanical failure compared to those of Col þ / þ /apoE À/À mice. Optical analysis further indicated altered collagen fiber orientation in the adventitia of Col R/R /apoE À/À mice. These results demonstrate that collagen content regulates aortic biomechanical properties and influences AAA formation. The pathogenesis of abdominal aortic aneurysm (AAA) remains uncertain, and the lack of definitive insight suggests a complex, multifactorial process. 1-4 AAA possesses major alterations in the content of extracellular matrix proteins: collagen and elastin. 3,[5][6][7] Interstitial collagen increases arterial stiffness, a property linked to AAA formation. 8,9 Clinical evidence links increased arterial stiffness or collagen content to aortic aneurysms, whereas nonaneurysmal portions of aortas in AAA patients have showed increased collagen accumulation. [10][11][12][13][14][15][16][17][18][19][20] However, whether collagen accumulation contributes causally to aneurysm formation or merely represents an epiphenomenon remains untested. To unravel the complex pathogenesis of AAA, we hypothesized that examination of both collagen content and its mechanical properties in mice with a genetic abnormality would provide novel insight.To test directly in vivo whether aortic stiffness due to excess collagen accumulation promotes aneurysm formation, we used collagenase-resistant knock-in (Col R/R ) mice. 21,22 This mouse strain bears a mutation in type I collagen, which comprises two-thirds of aortic collagen, at a cleavage site of the a1 (I) chain (Ile 776 to Pro 776 ) shared by collagenases of the matrix metalloproteinase (MMP) family (MMP-1/collagenase-1, MMP-8/collagenase-2 and MMP-13/collagenase-3). This mutation thus confers resistance to degradation, resulting in excess arterial collagen accumulation. 22 Our study used angiotensin II (AngII) infusion to provoke AAA formation in apolipoprotein E-deficient (apoE À/À ) mice as established by Daugherty et al. 23 AngII also stimulates collagen synthesis in vitro and induces collagen turnover and remode...

Abstract.Although serum deprivation induces apoptosis in several cell lines, biochemical characterization of the apoptosis in primary granulosa cells (GCs) induced by serum deprivation has rarely been reported. In the present study, GCs from small follicles of porcine ovaries were precultured under a serum-containing condition for seven days, then stepped down to a serum-free condition and cultured for the subsequent two days. GCs were subjected to DNA fragmentation and immunoblot analyses.Data indicated that serum deprivation induced GC apoptosis characterized by DNA laddering, which was associated with decreased expression of proliferating cell nuclear antigen (PCNA) and increased expression of p53 protein, Fas antigen and Fas ligand. Serum deprivation also resulted in an increase in a 115 kDa protein expression despite no detectable expression of a 66 kDa c-myc protein. This suggests that serum removal from primary GCs may activate multiple apoptotic pathways such as a p53-associated pathway and a Fas-Fas ligand pathway.

A cyclic depsipeptide, FR900359, isolated from Ardisia crenata was evaluated for vasorelaxant effects on rat aortic arteries. FR900359 caused concentration-dependent relaxation (1 nM-10 μM) in phenylephrine-precontracted endothelium-intact aortic rings, which was inhibited by addition of L-NMMA, a NOS inhibitor. In endothelium-denuded rings, the relaxant effect of low concentrations of FR900359 was diminished, but remained at high concentrations. In endothelium-denuded rings, FR900359 at 0.1 μM significantly attenuated high-K(+)-induced contractions and completely inhibited Ca(2+)-induced contractions. These results suggest that the vasorelaxant effect of FR900359 is mediated through the increased release of NO from endothelial cells at low concentrations, and can be attributed to inhibitory effects on voltage-dependent Ca(2+) channel- and receptor-operated Ca(2+) channel-dependent Ca(2+) influx at high concentrations.

Photoacoustic (PA) imaging has been considered an attractive imaging modality for sensitive and in-depth imaging of biomolecules with a high resolution in vivo. PA imaging probes utilizing fluorescence dyes, including indocyanine green (ICG), have been proposed to enhance PA signal intensity. On the other hand, nanomicelles modified with polysarcosine (PSar), a biocompatible hydrophilic polymer, on their surface have previously achieved rapid tumor uptake, suggesting active transport of PSar into tumor tissues. Thus, we hypothesized that PSar-based materials might be utilized as diagnostic probes for targeting tumors and therefore evaluated the potential of PSar labeled with an ICG derivative, ICG-PSar, as a PA imaging probe for targeting cancer. In this study, ICG-PSars with differing molecular weights (10, 20, and 30 kDa) were synthesized. In vitro cellular uptake studies using ICG-PSar demonstrated rapid uptake in colon26 tumor cells partially via macropinocytosis-mediated endocytosis. In vivo fluorescence imaging and biodistribution study indicated that ICG-PSar30k exhibited high accumulation in the tumor (8.4% dose/g), with high tumor-to-blood ratios reaching 4.6 at 24 h post injection of the probe. Finally, in vivo PA imaging studies showed that PA signal increased in tumors (251%) but not in blood vessels, achieving high contrast tumor imaging at 24 h after ICG-PSar30k probe injection. These results suggest that ICG-PSar has potential as a tumor-targeting PA imaging probe.

Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2 = 0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.