Immortalized human chondrocytes were established by transfection of primary cultures of juvenile costal chondrocytes with vectors encoding simian virus 40 large T antigen and selection in suspension culture over agarose. Stable cell lines were generated that exhibited chondrocyte morphology, continuous proliferative capacity (> 80 passages) in monolayer culture in serum-containing medium, and expression of mRNAs encoding chondrocyte-specific collagens II, IX, and XI and proteoglycans in an insulin-containing serum substitute. They did not express type X collagen or versican mRNA. These cells synthesized and secreted extracellular matrix molecules that were reactive with monoclonal antibodies against type II collagen, large proteoglycan (PG-H, aggrecan), and chondroitin-4-and chondroitin-6-sulfate. Interleukin-1,8 (IL-1p) decreased the levels of type II collagen mRNA and increased the levels of mRNAs for collagenase, stromelysin, and immediate early genes (egr-1, c-fos, c-jun, and jun-B). These cell lines also expressed reporter gene constructs containing regulatory sequences (-577/+3,428 bp) of the type II collagen gene (COL2A1) in transient transfection experiments, and IL-1p8 suppressed this expression by 50-80%. These results show that immortalized human chondrocytes displaying cartilage-specific modulation by IL-1,3 can be used as a model for studying normal and pathological repair mechanisms. (J. Clin. Invest. 1994. 94:2307-2316

This review focuses on the vascular smooth muscle cells present in the medial layer of the blood vessels wall in the fully differentiated state (dVSMCs). The dVSMC contractile phenotype enables these cells to respond in a highly regulated manner to changes in extracellular stimuli. Through modulation of vascular contractile force and vascular compliance dVSMCs regulate blood pressure and blood flow. The cellular and molecular mechanisms by which vascular smooth muscle contractile functions are regulated are not completely elucidated. Recent studies have documented a critical role for actin polymerization and cytoskeletal dynamics in the regulation of contractile function. Here we will review the current understanding of actin cytoskeletal dynamics and focal adhesion function in dVSMCs in order to better understand actin cytoskeleton connections to the extracellular matrix and the effects of cytoskeletal remodelling on vascular contractility and vascular stiffness in health and disease.

Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90-200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors.

We have investigated the functional relationship between metalloendopeptidase EC 3.4.24.15 (MP24.15) and the amyloid precursor protein involved in Alzheimer's disease (AD) and discovered that the enzyme promotes A␤ degradation. We show here that conditioned medium (CM) of MP24.15 antisense-transfected SKNMC neuroblastoma has significantly higher levels of A␤. Furthermore, synthetic-A␤ degradation was increased or decreased following incubation with CM of sense or antisense-transfected cells, respectively. Soluble A␤1-42 was degraded more slowly than soluble A␤1-40, while aggregated A␤1-42 showed almost no degradation. Alzheimer's disease (AD)1 is a progressive neurodegenerative disorder and the most common form of dementia in the elderly (1). Evidence indicates that accumulation of amyloid-␤ (A␤) deposits in senile plaques and in cerebrovasculature is associated with the pathophysiology of AD (2). The A␤ peptide is composed of 40 -42 amino acids (3). The events leading to its formation from the transmembrane amyloid precursor protein (APP) involve proteolytic cleavage by enzymes that have been termed: 1) ␤-secretase, which cleaves at the amino terminus of A␤, and 2) ␥-secretase, which cleaves at the carboxyl terminus. In the senile plaques A␤ is associated with a number of proteins, including ␣ 1 -antichymotrypsin (ACT) (4), which is a serine proteinase inhibitor of the serpin family as well as an acute-phase protein (5). Thus, we hypothesized 10 years ago that ACT may play a role in APP processing (4, 6).Soluble A␤ peptide can be detected in the conditioned media of a variety of cultured mammalian cells in vitro (7-9), as well as in serum and cerebrospinal fluid in vivo (10). The majority of secreted A␤ is 40 amino acids in length (A␤1-40), but approximately 10% of all A␤ is 42 amino acids in length (A␤1-42) (11). Little is known about how secreted A␤ is degraded and cleared from the extracellular milieu. The excessive cerebral accumulation of A␤ that occurs in AD could be explained in part by a decreased ability of the brain to degrade and clear A␤. If neural cells can be shown to release specific A␤-degrading proteinases, changes in the activity of such proteinases and/or their upregulation could represent a therapeutic approach to AD.We have explored the role of the metalloendopeptidase MP24.15 in the degradation of A␤. MP24.15 is a thiol-dependent enzyme that was purified to homogeneity from AD brain as a candidate ␤-secretase (12). McDermot et al. (13) also identified a partially purified MP24.15 as a potential ␤-secretase using synthetic peptide substrates. In vitro, MP24.15 has been shown to be involved in the inactivation of a number of neuropeptides, including somatostatin, bradykinin, substance P, and neurotensin (14 -16). The cDNA coding for MP24.15 was subsequently cloned from a human brain library (17,18). In an attempt to further examine the ␤-secretase properties of MP24.15 under physiologic conditions, we transfected human neuroblastoma cells with MP24.15 cDNA in the sense and antisense directi...

The amyloid precursor protein (APP) must fulfill important roles based on its sequence conservation from fly to human. Although multiple functions for APP have been proposed, the best-known role for this protein is as the precursor of Abeta peptide, a neurotoxic 39-43-amino acid peptide crucial to the pathogenesis of Alzheimer's disease. To investigate additional roles for APP with an eye toward understanding the molecular basis of the pleiotropic effects ascribed to APP, we isolated proteins that interacted with the plasma membrane isoform of APP. We employed a membrane-impermeable crosslinker to immobilize proteins binding to transmembrane APP in human embryonic kidney (HEK)293 cells expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identified as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopurified from neurons. To confirm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full-length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and confirmed our initial findings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular cross talk to modulate cell function. Finally, cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identification of new functions for APP.