Role of discoidin domain receptors 1 and 2 in human smooth muscle cell-mediated collagen remodeling: potential implications in atherosclerosis and lymphangioleiomyomatosis

Obstructive diseases of blood vessels and the lung are characterized by degradation and synthesis of new extracellular matrix (ECM) components. Regulated remodeling of the ECM in diseases such as atherosclerosis and lymphangioleiomyomatosis (LAM), both characterized by excessive accumulation of smooth muscle cells (SMCs), is thought to be controlled in part by cell surface receptors for specific ECM components. Discoidin domain receptors (DDR) 1 and 2 represent a family of tyrosine kinase collagen receptors that are activated by fibrillar collagens. To test the hypothesis that DDR may be involved in ECM remodeling by SMCs in vivo, we analyzed DDR expression by reverse transcriptase-polymerase chain reaction and immunohistochemistry and demonstrate that both DDR1 and DDR2 are up-regulated in nodules of LAM as compared to normal controls, and are expressed in lesions of atherosclerosis. In vitro, retroviral overexpression of DDR1 or DDR2 in human SMCs cultured on polymerized collagen gels leads to a reduction of collagen expression and induces matrix metalloproteinase (MMP) 1 at both mRNA and protein levels, but only DDR2 enhances MMP2 activation. Moreover, DDR2 overexpression increases SMC-mediated collagen and elastin degradation in vitro. Using laser microdissection, we extend our studies to the analysis of SMCs from LAM nodules where we observe higher MMP1 expression and MMP2 activation. Taken together, these data provide evidence for the potential roles of DDR1 and DDR2 in the regulation of collagen turnover mediated by SMCs in obstructive diseases of blood vessels and the lung

Simvastatin reduces MMP1 expression in human smooth muscle cells cultured on polymerized collagen by inhibiting Rac1 activation

OBJECTIVE: Activation of collagen receptors expressed by smooth muscle cells induces matrix metalloproteinase (MMP) expression. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to interfere with integrin signaling, but their effects on collagen receptor-mediated MMP expression have not been investigated. METHODS AND RESULTS: In the present study, we show that simvastatin (3 micromol/L) reduces MMP1 expression and secretion in human smooth muscle cells cultured on polymerized type I collagen by 39.9+/-11.2% and 36.0+/-2.3%, respectively. Reduced MMP1 protein levels correlate with a similar decrease in MMP1 promoter activity (-33.0+/-8.9%), MMP1 mRNA levels (-37.8+/-10.5%), and attenuation of smooth muscle cell collagen degradation (-34.2+/-6.1%). Mevalonate, and the isoprenoid derivative geranylgeraniol, precursors of geranylgeranylated proteins, completely prevent the inhibitory effect of simvastatin on MMP1. Moreover, the protein geranylgeranyltransferase inhibitor GGTI-286 significantly decreases MMP1 expression. Retroviral overexpression of dominant-negative mutants of geranylgeranylated Rac1 lead to a reduction of MMP1 protein (-50.4+/-5.4%) and mRNA levels (-97.9+/-1.0%), and knockdown of Rac1 by small interfering RNA downregulates MMP1 expression. Finally, simvastatin reduces GTP-bound Rac1 expression levels in smooth muscle cells cultured on polymerized collagen. CONCLUSIONS: These results demonstrate that simvastatin, by inhibiting Rac1 activity, reduces MMP1 expression and collagen degradation in human smooth muscle cells

Cleavage of focal adhesion kinase in vascular smooth muscle cells overexpressing membrane-type matrix metalloproteinases

Membrane-type matrix metalloproteinases (MT-MMPs) were initially identified as cell surface activators of MMP-2 (gelatinase A). We have reported that MT1-MMPs and MT3-MMPs are expressed by activated vascular smooth muscle cells (SMCs) and play a role in the regulation of cell function. Overexpression of MT-MMPs results in cell rounding, decreased adherence, and increased migration. Because integrin-mediated cell adhesion regulates these events, we have investigated the functional relationship between MT-MMPs and focal adhesion assembly

Precision measurement of the deuteron spin structure function g1dg^{d}_{1}

We report on a high-statistics measurement of the deuteron spin structure function g[sup d][sub 1] at a beam energy of 29 GeV in the kinematic range 0.029 < x < 0.8 and 1 < Q2 < 10 (GeV/c)2. The integral Gamma [sup d][sub 1] = (integral)[sup 1][sub 0]g[sup d][sub 1]dx evaluated at fixed Q2 = 3 (GeV/c)2 gives 0.042 ± 0.003(stat) ± 0.004(syst). Combining this result with our earlier measurement of g[sup p][sub 1], we find Gamma [sup p][sub 1]- Gamma [sup n][sub 1] = 0.163 ± 0.010(stat) ± 0.016(syst), which agrees with the prediction of the Bjorken sum rule with O( alpha [sup 3][sub s]) corrections, Gamma [sup p][sub 1]- Gamma [sup n][sub 1] = 0.171 ± 0.008. We find the quark contribution to the proton helicity to be Delta q = 0.30 ± 0.06

Measurements of R=sigma_L/sigma_T for 0.03<x<0.1 and Fit to World Data

Measurements were made at SLAC of the cross section for scattering 29 GeV electrons from carbon at a laboratory angle of 4.5 degrees, corresponding to 0.03<x<0.1 and 1.3<Q^2<2.7 GeV^2. Values of R=sigma_L/sigma_T were extracted in this kinematic range by comparing these data to cross sections measured at a higher beam energy by the NMC collaboration. The results are in reasonable agreement with pQCD calculations and with extrapolations of the R1990 parameterization of previous data. A new fit is made including these data and other recent results.Comment: 8 pages, 4 figures, late

Next-to-Leading Order QCD Analysis of Polarized Deep Inelastic Scattering Data

We present a Next-to-Leading order perturbative QCD analysis of world data on the spin dependent structure functions $g_1^p, g_1^n$, and $g_1^d$, including the new experimental information on the $Q^2$ dependence of $g_1^n$. Careful attention is paid to the experimental and theoretical uncertainties. The data constrain the first moments of the polarized valence quark distributions, but only qualitatively constrain the polarized sea quark and gluon distributions. The NLO results are used to determine the $Q^2$ dependence of the ratio $g_1/F_1$ and evolve the experimental data to a constant $Q^2 = 5 GeV^2$. We determine the first moments of the polarized structure functions of the proton and neutron and find agreement with the Bjorken sum rule.Comment: 21 pages, 4 figures; final version to be published in Phys. Lett. B. References updated. Uses elsart.cls version 1996/04/22, 2e-1.4

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed

Cleavage of focal adhesion kinase in vascular smooth muscle cells overexpressing membrane-type matrix metalloproteinases

none6siMembrane-type matrix metalloproteinases (MT-MMPs) were initially identified as cell surface activators of MMP-2 (gelatinase A). We have reported that MT1-MMPs and MT3-MMPs are expressed by activated vascular smooth muscle cells (SMCs) and play a role in the regulation of cell function. Overexpression of MT-MMPs results in cell rounding, decreased adherence, and increased migration. Because integrin-mediated cell adhesion regulates these events, we have investigated the functional relationship between MT-MMPs and focal adhesion assembly.noneT. Shofuda; K. Shofuda; N. Ferri; R.D. Kenagy; E.W. Raines; A.W. ClowesT., Shofuda; K., Shofuda; Ferri, Nicola; R. D., Kenagy; E. W., Raines; A. W., Clowe