Human bone marrow was harvested by means of iliac crest aspiration and cultured under conditions that promote an osteoblastic phenotype. Human bone marrow aspirates from 30 normal subjects, ages 8-80 years, with no systemic illness, yielded a mean of 92 +/- 65 x 10(6) nucleated cells per 2 ml of aspirate. The prevalence of potential osteoblastic progenitors was estimated by counting the number of alkaline phosphatase-positive colonies. This assay demonstrated a mean of 43 +/- 28 alkaline phosphatase-positive colonies per 10(6) nucleated cells, which was about one per 23,000 nucleated cells. The prevalence of these colonies was positively correlated with the concentration of nucleated cells in the original aspirate (p = 0.014) and was negatively correlated with donor age (p = 0.020). The population of alkaline phosphatase-positive colonies in this model sequentially exhibited markers of the osteoblastic phenotype; essentially all colonies (more than 99%) stained positively for alkaline phosphatase on day 9. Matrix mineralization, which was associated with the synthesis of bone sialoprotein, was demonstrated on day 17 with alizarin red S staining. On day 45, cells that were stimulated with 1,25-dihydroxyvitamin D3 synthesized and secreted osteocalcin at concentrations consistent with known osteoblastic cell lines. This model provides a useful method for the assay of progenitors of connective tissue from human subjects, examination of the effects of aging and selected disease states on this progenitor population, and investigation into the regulation of human osteoblastic differentiation.
An elevated concentration of plasma total homocysteine is an independent risk factor for cardiovascular disease. Greater than 80% of circulating homocysteine is covalently bound to plasma protein by disulfide bonds.
Autosomal recessive and autosomal dominant forms of WeillMarchesani syndrome, an inherited connective tissue disorder, are caused by mutations in ADAMTS10 (encoding a secreted metalloprotease) and FBN1 (encoding fibrillin-1, which forms tissue microfibrils), respectively, yet they are clinically indistinguishable. This genetic connection prompted investigation of a potential functional relationship between ADAMTS10 and fibrillin-1. Specifically, fibrillin-1 was investigated as a potential ADAMTS10 binding partner and substrate, and the role of ADAMTS10 in influencing microfibril biogenesis was addressed. Using ligand affinity blotting and surface plasmon resonance, recombinant ADAMTS10 was found to bind to fibrillin-1 with a high degree of specificity and with high affinity. Two sites of ADAMTS10 binding to fibrillin-1 were identified, one toward the N terminus and another in the C-terminal half of fibrillin-1. Confocal microscopy and immunoelectron microscopy localized ADAMTS10 to fibrillin-1-containing microfibrils in human tissues. Furin-activated ADAMTS10 could cleave fibrillin-1, but innate resistance of ADAMTS10 zymogen to propeptide excision by furin was observed, suggesting that, unless activated, ADAMTS10 is an inefficient fibrillinase. To investigate the role of ADAMTS10 in microfibril biogenesis, fetal bovine nuchal ligament cells were cultured in the presence or absence of ADAMTS10. Exogenously added ADAMTS10 led to accelerated fibrillin-1 microfibril biogenesis. Conversely, fibroblasts obtained from a Weill-Marchesani syndrome patient with ADAMTS10 mutations deposited fibrillin-1 microfibrils sparsely compared with unaffected control cells. Taken together, these findings suggest that ADAMTS10 participates in microfibril biogenesis rather than in fibrillin-1 turnover.Inherited connective tissue disorders that result from mutations of genes encoding extracellular matrix (ECM) 3 components or their modifying enzymes are potentially insightful in identifying structural and regulatory roles of ECM constituents or their supramolecular assemblies. A classic example of such an ECM component is fibrillin-1, a large, secreted glycoprotein, which is a major component of 8 -12-nm-diameter tissue microfibrils (1). Bundles of microfibrils, containing fibrillin-1, fibrillin-2, and fibrillin-3 (in humans), are ubiquitous in the body but are especially abundant in tissues containing elastin (2). The structural role of microfibrils includes maintenance of the ocular lens in its appropriate position in the eye via the zonule of Zinn (the suspensory ligament of the lens), which comprises primarily fibrillin-1 microfibrils (2, 3). Inadequacy of the zonule or traumatic breakage of zonule fibers can result in subluxation or dislocation of the lens (ectopia lentis). Dominantly inherited FBN1 mutations lead to diverse connective tissue anomalies, the most common of which is Marfan syndrome (MFS) (4). Major features of MFS include musculoskeletal anomalies, aortic aneurysms, and ectopia lentis. Several features of MFS appear...
There is mounting evidence that perturbations in endoplasmic reticulum (ER) function play a key role in the pathogenesis of a broad range of diseases. We have examined the ability of ER stress to modulate leukocyte binding to colonic and aortic smooth muscle cells. In vitro, control smooth muscle cells bind few leukocytes, but treatment with compounds that induce ER stress, including tunicamycin, A23187, and thapsigargin, promotes leukocyte binding. Likewise, dextran sulfate, another agent capable of inducing ER stress and promoting inflammation in vivo, strongly induces leukocyte adhesion. The bound leukocytes are released by hyaluronidase treatment, indicating a critical role for hyaluronan-containing structures in mediating leukocyte binding. Affinity histochemistry demonstrated that hyaluronan accumulates and is present in cable-like structures in the treated, but not the untreated, cultures and that these structures serve as attachment sites for leukocytes. Hyaluronan-rich regions of both murine and human inflamed colon contain numerous cells that stain intensely for ER-resident chaperones containing the KDEL sequence, demonstrating a relationship between ER stress and hyaluronan deposition in vivo. These results indicate that ER stress may contribute to chronic inflammation by forming a hyaluronan-rich extracellular matrix that is conducive to leukocyte binding.
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