Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition.

Cadherin-13 (CDH13), a unique glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules, has been identified as a risk gene for attention-deficit/hyperactivity disorder (ADHD) and various comorbid neurodevelopmental and psychiatric conditions, including depression, substance abuse, autism spectrum disorder and violent behavior, while the mechanism whereby CDH13 dysfunction influences pathogenesis of neuropsychiatric disorders remains elusive. Here we explored the potential role of CDH13 in the inhibitory modulation of brain activity by investigating synaptic function of GABAergic interneurons. Cellular and subcellular distribution of CDH13 was analyzed in the murine hippocampus and a mouse model with a targeted inactivation of Cdh13 was generated to evaluate how CDH13 modulates synaptic activity of hippocampal interneurons and behavioral domains related to psychopathologic (endo)phenotypes. We show that CDH13 expression in the cornu ammonis (CA) region of the hippocampus is confined to distinct classes of interneurons. Specifically, CDH13 is expressed by numerous parvalbumin and somatostatin-expressing interneurons located in the stratum oriens, where it localizes to both the soma and the presynaptic compartment. Cdh13(-/-) mice show an increase in basal inhibitory, but not excitatory, synaptic transmission in CA1 pyramidal neurons. Associated with these alterations in hippocampal function, Cdh13(-/-) mice display deficits in learning and memory. Taken together, our results indicate that CDH13 is a negative regulator of inhibitory synapses in the hippocampus, and provide insights into how CDH13 dysfunction may contribute to the excitatory/inhibitory imbalance observed in neurodevelopmental disorders, such as ADHD and autism.

T-cadherin loss induces an invasive phenotype in human keratinocytes and squamous cell carcinoma (SCC) cells in vitro and is associated with malignant transformation of cutaneous SCC in vivo.

BACKGROUND: Cadherins play important roles in controlling keratinocyte growth, differentiation and survival. Atypical glycosylphosphatidylinositol-anchored T-cadherin (T-cad) is highly expressed in the basal keratinocyte layer of skin. The role of T-cad in keratinocyte biology and pathology is unclear. OBJECTIVES: To define the role of T-cad in the pathogenesis of cutaneous squamous cell carcinoma (SCC) through gain-of-function and loss-of-function studies in vitro and through examination of T-cad expression patterns in human cutaneous SCC specimens in relation to histological classification of degree of tumour differentiation. METHODS: In vitro studies employed lentiviral-mediated overexpression/silencing of T-cad in normal human keratinocyte (HaCaT) and SCC (A431) cell lines, monolayer and multicellular spheroid culture models, cell morphology analyses and assays of random motility and invasion. Immunohistochemistry was performed on skin specimens from patients with actinic keratosis, Bowen disease or SCC. RESULTS: In vitro, silencing of T-cad induced a morphologically elongated and disorganized cell phenotype, increased random motility and markedly enhanced invasive potential. Overexpression of T-cad induced a morphologically spread and compact cell phenotype and blunted invasive potential. In vivo, regional loss of T-cad expression was more frequent and prominent in SCC classified as moderately-to-poorly differentiated than in SCC classified as well differentiated. However, in both categories aberrant and/or absence of T-cad expression was associated with histological features of a potentially more malignant and invasive phenotype of cutaneous SCC. CONCLUSIONS: T-cad is a controlling determinant of SCC phenotype and invasive behaviour and its loss is associated with the process of malignant transformation from noninvasive to invasive SCC.

Monocyte integrin expression and monocyte-platelet complex formation in humans with coronary restenosis.

1. In the present study, we sought to determine whether patients with restenosis after coronary stenting possess increased monocyte reactivity, as manifested by a higher level of adhesion molecule expression and an enhanced propensity to form monocyte-platelet aggregates after activation in vitro. 2. Anti-coagulated peripheral venous blood from 24 patients, 10 with and 14 without angiographically verified restenosis, was obtained. Leucocyte antigen expression and the number of leucocyte-platelet complexes were measured by flow cytometry after activation in whole blood. 3. Surface integrin Mac-1 (CD11b/CD18) and VLA-4 (CD49d/ CD29) expression on monocytes and the relative number of monocyte-platelet complexes after in vitro activation were significantly elevated in patients with restenosis compared with patients without restenosis (fluorescence intensities of 1425 +/- 76 vs 1195 +/- 71, 87 +/- 7 vs 65 +/- 6 and 47 +/- 4 vs 29 +/- 3% for for Mac-1, VLA-4 and monocyte-platelet complexes, respectively; P < 0.05 for each parameter). 4. The results suggest that restenosis is associated with increased monocyte VLA-4 and Mac-1 integrin expression and monocyte-platelet complex formation, which can be revealed after activation in vitro.

LDL binds to surface-expressed human T-cadherin in transfected HEK293 cells and influences homophilic adhesive interactions.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.

Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms.

In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. We conclude that hSMC migration in response to uPA depends upon on its binding to uPAR, whereas uPA-stimulated DNA synthesis in these cells requires proteolysis and plasmin generation.

Urokinase plasminogen activator system in humans with stable coronary artery disease.

1. The present study compares plasma urokinase plasminogen activator (uPA) peptide levels, plasma plasminogen inhibitor (PAI-1) activity and urokinase receptors (uPAR) on peripheral blood monocytes of patients with stable coronary artery disease (SCAD) and healthy volunteers. 2. Urokinase plasminogen activator levels were analysed by ELISA and PAI-1 activity was determined by a plasmin generation method using the chromogenic substrate S2390. Relative uPAR numbers and the adhesion molecules CD11b/CD18 on peripheral blood monocytes were estimated using specific antibodies and flow cytometry. 3. Patients with SCAD were found to have higher plasma uPA peptide levels than age-matched healthy subjects (10.40 +/- 0.99 vs 8.25 +/- 0.53 pmol/L, respectively; P < 0.05). 4. Plasma PAI-1 activity was also higher in patients with SCAD than in healthy subjects (13.6 +/- 2.5 vs 5.2 +/- 1.0 IU/mL, respectively; P < 0.05). 5. Relative uPAR and CD11b/CD18 adhesion molecules were similar on peripheral blood monocytes of patients with SCAD and in healthy subjects. 6. The data indicate a pattern of expression/activity of uPA and PAI-1 in patients with SCAD suggestive of an impaired fibrinolytic ability.

Effect of Ox-LDL on endothelium-dependent response in pig ciliary artery: prevention by an ET(A) antagonist.

PURPOSE: To investigate whether oxidized low-density lipoprotein (Ox-LDL) affects endothelium-dependent responses in isolated porcine ciliary arteries. METHODS: In a myograph system for isometric force measurements, quiescent vessels were incubated with 50 microg/ml, 100 microg/ml, or 200 microg/ml Ox-LDL; 100 microg/ml native LDL (n-LDL); 1 microM of the ET(A)- endothelin receptor antagonist BQ 123; 100 microg/ml Ox-LDL coadministered with 1 microM BQ 123; or 100 microg/ml Ox-LDL coadministered with 50 microM of the protein synthesis inhibitor cycloheximide. Vessels with nonfunctional endothelium (intentionally and mechanically damaged) were also exposed to 100 microg/ml Ox-LDL. Two hours later, vessels were washed, precontracted with the thromboxane A2 analog U 46619 (approximately 0.1 microM), and exposed to bradykinin (0.1 nM to 3 microM), an endothelium-dependent relaxing agent. RESULTS: In quiescent vessels, Ox-LDL evoked delayed contractions. In contrast, no contractions were observed after exposure to n-LDL, BQ 123, Ox-LDL with BQ 123, or Ox-LDL with cycloheximide. In vessels with nonfunctional endothelium, Ox-LDL did not evoke contraction. Bradykinin-induced relaxations were inhibited in a dose-dependent manner by Ox-LDL, but not by n-LDL, BQ 123 alone, Ox-LDL with BQ 123, or Ox-LDL with cycloheximide. CONCLUSIONS: In porcine ciliary arteries, Ox-LDL affects endothelium-dependent responses through the activation of ET(A)- endothelin receptors. As Ox-LDL can accumulate in atherosclerotic plaques, such a mechanism might be involved in the occlusion of the ophthalmic circulation observed in patients with hypercholesterolemia and atherosclerosis.

Identification of 130 kDa cell surface LDL-binding protein from smooth muscle cells as a partially processed T-cadherin precursor.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.

Density- and proliferation status-dependent expression of T-cadherin, a novel lipoprotein-binding glycoprotein: a function in negative regulation of smooth muscle cell growth?

The atypical low density lipoprotein (LDL) binding proteins (Mr 105 and 130 kDa; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T-cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T-cadherin and the density and proliferation status of SMC. T-cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand-binding techniques. T-cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T-cadherin levels were reversibly modulated by switching cultures between serum-free (upmodulation) and serum-containing (downmodulation) conditions. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF) or insulin-like growth factor (IGF) elicited a dose- and time-dependent downmodulation that was reversible after transfer of SMC to growth factor-free medium. Our results support the hypothesis that T-cadherin may function as a negative determinant of cell growth.

T-cadherin and signal-transducing molecules co-localize in caveolin-rich membrane domains of vascular smooth muscle cells.

Cadherins are a family of cellular adhesion proteins mediating homotypic cell-cell binding. In contrast to classical cadherins, T-cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T-cadherin might function as a signal-transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X-100 solubilization and density gradient centrifugation techniques. We demonstrate that T-cadherin is enriched in a minor detergent-insoluble low-density membrane domain and co-distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI-anchored proteins (CD-59, uPA receptor) and signal-transducing molecules (G alpha s protein and Src-family kinases), but completely excluded cell-cell and cell-matrix adhesion molecules (N-cadherin and beta1-integrin). Coupling of T-cadherin with signalling molecules within caveolae might enable cellular signal transduction.

Characteristics of smooth muscle cell lipoprotein binding proteins (p105/p130) as T-cadherin and regulation by positive and negative growth regulators.

Smooth muscle cells (SMC) express atypical surface low density lipoprotein (LDL) binding proteins of M(r)105 and M(r)130 (p105 and p130) which have been putatively identified as the cell adhesion glycoprotein T-cadherin. Using cultured human and rat aortic SMC and analysis by ligand (LDL)- and immuno-blotting techniques we now confirm identity of p105 and p130 as T-cadherin, as adjudged by sensitivity to PI-PLC cleavage, insensitivity to trypsin degradation in the presence of calcium, and immunoreactivity to anti-T-cadherin peptide antisera. The function of T-cadherin (p105/p130) in the vasculature is unknown. The proteins were downmodulated by the peptide growth factors PDGF-BB, IGF, EGF, and bFGF, but not by vasoactive peptide hormones (angiotensin II, vasopressin, bradykinin, and endothelin). TGF beta, a recognized inhibitor of SMC proliferation, per se had no effect but inhibited growth factor-induced p105/p130 downmodulation. Expression of p105/p130 in quiescent SMC and growth-stimulated SMC (respectively, in serum-free and serum or PDGF-BB containing culture conditions) was increased by forskolin and 8-Br-cyclic GMP, both anti-mitogenic substances, but was unaffected by phorbol ester, calcium ionophores, or calcium antagonists. The findings are compatible with a function for the lipoprotein binding proteins (T-cadherin) in negative regulation of SMC growth.

Identification of an atypical lipoprotein-binding protein from human aortic smooth muscle as T-cadherin.

We have previously described an atypical lipoprotein-binding protein of about 105 kDa (p105) in membranes of vascular smooth muscle cells (VSMCs) that is distinct from currently known lipoprotein receptors. In the present work we have developed a procedure for purification of p105 from human aortic media. Partial sequencing of purified protein has revealed identity of p105 with human T-cadherin. Anti-peptide antisera raised against human T-cadherin recognized a protein spot corresponding to the purified p105 on two-dimensional Western blots. The antisera also inhibited LDL binding to p105 on ligand blots. We conclude that the 105 kDa lipoprotein-binding protein present in human VSMCs is T-cadherin, an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell-cell adhesion proteins.

Molecular mechanism of the desensitization of beta-adrenergic receptors and adenilate cyclase by hypoxia in human endothelial cells.

Long-term oxygen deficiency in vivo leads to the progressive blunting of responsiveness to sympathetic stimulation and blood catecholamines in many human and animal tissues. In order to better understand the molecular processes that underlie this phenomenon we examined the effect of hypobaric hypoxia (290 mm Hg, pO2 = 40 mM Hg) on the--beta-adrenoreceptor (beta-AR) density and the activity of adenylate cyclase (AC) and phosphoinositide turnover (PI-turnover) in cultures of human pulmonary artery and umbilical vein cells. We discovered that 30 min of hypobaric hypoxia increased basal levels of inositol mono-, bis- and tris-phosphate, products of PI-turnover in endothelial cells (EC). After 60 min of hypoxia their content amounted to 250-300% of the basal level. Desensitization of PI-turnover to histamine stimulation in EC was observed after 60 min of hypoxia. Basal and isoproterenol (beta-AR-agonist)-stimulated AC activities therewith were markedly reduced. beta-AR-density was decreased in EC membranes after 2-3 hrs of hypoxia. Similar desensitization of beta-AR and AC occurred after 1-2 hrs treatment of EC with histamine and platelet activating factor (stimulators of PI-turnover) and with phorbol myristate acetate (PK C activator). Neither hyproxia nor phorbol myristate acetate influenced beta-AR density or AC activity in protein kinase C-deficient EC (72 hrs treatment with phorbol myristate acetate). The data suggest that hypoxia-induced desensitization of beta-AR and AC in endothelial cells is mediated via hypozia-stimulated turnover and subsequent protein kinase C activation.

Ligand selectivity of 105 kDa and 130 kDa lipoprotein-binding proteins in vascular-smooth-muscle-cell membranes is unique.

Using ligand blotting techniques, with low-density lipoprotein (LDL) as ligand, we have previously described the existence of atypical lipoprotein-binding proteins (105 kDa and 130 kDa) in membranes from human aortic medical tissue. The present study demonstrates that these proteins are also present in membranes from cultured human (aortic and mesenteric) and rat (aortic) vascular smooth-muscle cells (VSMCs). To assess the relationship of 105 and 130 kDa lipoprotein-binding proteins to known lipoprotein receptors, ligand binding specificity was studied. We tested effects of substances known to antagonize ligand binding to either the LDL [apolipoprotein B,E (apo B,E)] receptor (dextran sulphate, heparin, pentosan polysulphate, protamine, spermine, histone), the scavenger receptor (dextran sulphate, fucoidin), the very-low-density-lipoprotein (VLDL) receptor [receptor-associated protein (RAP)], or LDL receptor-related protein (RAP, alpha 2-macroglobulin, lipoprotein lipase, exotoxin-A). None of these substances, with the exception of dextran sulphate, influenced binding of LDL to either 105 or 130 kDa proteins. Sodium oleate or oleic acid, known stimuli for the lipoprotein binding activity of the lipolysis-stimulated receptor, were also without effect. LDL binding to 105 and 130 kDa proteins was inhibited by anti-LDL (apo B) antibodies. LDL and VLDL bound to 105 and 130 kDa proteins with similar affinities (approximately 50 micrograms/ml). The unique ligand selectivity of 105 and 130 kDa proteins supports the existence of a novel lipoprotein-binding protein that is distinct from all other currently identified LDL receptor family members. The similar ligand selectivity of 105 and 130 kDa proteins suggests that they may represent variant forms of an atypical lipoprotein-binding protein.

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects.

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.

Endothelium-modulated proliferation of medial smooth muscle cells: influence of angiotensin II and converting enzyme inhibition.

This study investigated the role of the endothelium and angiotensin II (Ang II) in regulating medial smooth muscle cell (SMC) proliferation. [3H]-thymidine incorporation into medial SMC of rat arteries was examined in vivo, using ballooned rat carotid arteries, as well as in vitro, using cultures of aortic tissue rings (organoids). In vivo, maximal medial [3H]-thymidine incorporation occurred within 3 days post-ballooning. In endothelium-denuded organoids, maximum medial DNA synthesis was achieved after 7 days of culture. [3H]-thymidine-labelling of SMC in intact organoids (with endothelium) increased minimally during culture, indicating that the endothelium provided protection with respect to medial proliferation under basal conditions (culture in the presence of 1% plasma-derived serum). Inclusion of 10(-7) M Ang II significantly elevated medial [3H]-thymidine incorporation above that in control cultures. The stimulatory effect of Ang II was much more pronounced in intact organoids that in endothelium-denuded organoids, indicating synergistic growth regulation by Ang II and endothelium-derived factors. When organoids were cultured in the combined presence of Ang II and the ACE inhibitor cilazaprilat, labelling indices of intact organoids were also significantly increased above control, but to a lower level than those obtained in the presence of Ang II alone. However, for endothelium-denuded organoids, medial [3H]-thymidine incorporation in the combined presence of Ang II and cilazaprilat was not significantly different from that in untreated controls. Thus, cilazaprilat exerts both endothelium-dependent and endothelium-independent negative regulatory effects on medial SMC proliferation.

Functional aspects of vascular tenascin-C expression.

The arterial tenascin C expression in vivo and in vitro has been studied using immunohistochemistry. The functional relevance of localized tenascin C expression was assessed in vitro using various human cell types involved in the progression of vascular disease. Normotensive and hypertensive rats exhibited age-dependent patterns of vascular (aorta) tenascin expression, but the lumen-to-media-directed progression of tenascin induction was accelerated in hypertensive rats. Tenascin-rich neointimal lesions (spontaneous) were observed at branching sites of aorta from aged (80 weeks) hypertensive rats. Subendothelial tenascin foci contained lipid-laden smooth muscle cells and monocytes/macrophages. Medial tenascin foci encaged smooth muscle cells which synthesized DNA. Tenascin was expressed both in vivo and in vitro by endothelial and smooth muscle cells but not by monocytes/macrophages; angiotensin II, oxidized-low density lipoprotein and transforming growth factor beta 1 induced expression of tenascin transcripts and glycoprotein in vitro. Endothelial and smooth muscle cells, but not monocytes, adhered to tenascin substrata. Tenascin reduced focal adhesion integrity in confluent endothelial and smooth muscle cell cultures. Angiotensin II-induced migration of endothelial and smooth muscle cells was accompanied by tenascin deposition within extracellular matrix migration trails. Tenascin may function both as a defense against monocyte invasion and medial smooth muscle replication, as well as a substratum for directed endothelial and smooth muscle cell migration.

Stretch affects phenotype and proliferation of vascular smooth muscle cells.

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state.

Atypical low density lipoprotein binding site that may mediate lipoprotein-induced signal transduction.

The characteristics of low density lipoprotein (LDL) binding in quiescent cultures of human vascular smooth muscle cells (VSMC) have been further investigated and compared with the characteristics of high affinity LDL binding in human fibroblasts [via the apolipoprotein (apo) B/E receptor] and with the properties of LDL-induced phosphoinositide catabolism in VSMC. In VSMC the bulk of specific 125I-LDL binding occurs at a low affinity site, several characteristics of which are distinct from those of 125I-LDL binding to the apo B/E receptor in fibroblasts. (a) The affinity of LDL binding in VSMC is 25-50 times lower than that in fibroblasts (Kd approximately 50 micrograms/ml versus Kd approximately 2 micrograms/ml). (b) The kinetics of LDL association and dissociation in VSMC are more rapid than those in fibroblasts. (c) In contrast to apo B/E receptor-mediated binding of LDL in fibroblasts, binding of LDL to VSMC is insensitive to heparin, chemical modification of lysine residues, and chelation (with EDTA) of divalent cations. (d) Apo E-free high density lipoprotein 3 displaces labeled LDL more effectively in VSMC than in fibroblasts. (e) The ratio of bound/internalized LDL to degraded LDL differs markedly between fibroblasts and VSMC. LDL-stimulated phosphoinositide catabolism in VSMC, which occurs with an activation constant similar to the Kd for low affinity LDL binding, is insensitive to heparin, modification of lysine and arginine residues in LDL, and chelation of divalent cations. Thus, the atypical low affinity receptor in these cells may mediate the effects of LDL on signal transduction.