David M. Larson

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Gap junction messenger RNA expression by vascular wall cells.

Gap junctions between vessel wall cells provide a pathway for the intercellular exchange of ions and small molecules. Pure cultures of microvascular and macrovascular endothelial and smooth muscle cells, vascular pericytes, and several nonvascular cell lines were tested for junctional communication by fluorescent dye transfer. All of the vascular wall cells were capable of dye transfer. Since gap junctions are formed by a family of related proteins (connexins) whose unique domains may confer physiological regulatory properties, we tested total RNA from these cultures by Northern blot analysis for expression of the currently available, characterized, and cloned mammalian gap junction proteins: connexin26, connexin32, and connexin43. All of the vascular wall cells expressed connexin43 messenger RNA. Connexin43 was expressed in vascular cells from bovine, porcine, rat, and human sources. Several nonvascular cell lines of mesenchymal origin also expressed connexin43 messenger RNA. When high stringency Northern blots were used, messenger RNAs for connexin32 or connexin26 were not detected in any of the vascular wall cells but were expressed in several cell lines of epithelial origin. Freshly isolated and purified aortic endothelial and smooth muscle RNA preparations similarly contained only connexin43 messenger RNA, excluding the possibility of culture-induced alterations in gene expression. The expression of connexin43 by all vascular wall cells may provide a mechanism for the functional integration of the vessel wall by gap junctions.

Junctional transfer of small molecules in cultured bovine brain microvascular endothelial cells and pericytes

We have utilized cultures of bovine brain microvascular endothelial cells (MEC) and pericytes to study two aspects of intercellular relations in the microvasculature. First, the apparent contradiction between the reported demonstration of dye transfer between endothelial cells in capillaries and venules in rat omentum and the lack of ultrastructurally demonstrable interendothelial gap junctions in the same vessels in omentum, brain, and other tissues led us to examine this problem in vitro. MEC showed extensive transfer of both fluorescent dye (Lucifer yellow CH, 96% transfer incidence in primary culture) and radiolabeled uridine nucleotides (97%). Freeze-fracture replicas of MEC revealed both gap and tight junctions. These results demonstrate that MEC are capable of producing gap junctions and engaging in junctional communication in vitro. Second, we have examined the interaction of pericytes with MEC. Cultured pericytes showed gap junctions in freeze-fracture replicas, variable dye transfer (cell density dependent, 19-91%), and extensive nucleotide transfer (94%). While the incidence of dye transfer between MEC and pericytes was low (10-31%), nucleotide transfer between these cells was extensive (86-96%). The demonstration of junctional transfer between MEC and pericytes in vitro may be particularly significant considering the high frequency of junctional contact between these cells in vivo. These cultured cell models should help us to better understand the complex interactions of vessel wall cells in microvascular physiology and pathophysiology.

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

SummaryGap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

SummaryHuman arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ss1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Activation of Smooth Muscle Cell Outgrowth From BB/Wor Rat Aortas

An essential event in atherogenesis is the migration and proliferation of vascular smooth muscle cells (VSMCs), which contributes to the fibrocellular component of the atherosclerotic intimai thickening. We have been modeling this process by studying the outgrowth of VSMC from aortic expiants onto tissue-culture plastic substrate. Our hypothesis is that certain risk factors for atherosclerosis favor increases in subpopulations of cells with enhanced responsiveness to a variety of migratory and proliferative stimuli, in vivo and in vitro. As a known risk factor for atherosclerosis, diabetes might be reasonably postulated to induce such cell populations with altered susceptibility to additional noxious stimuli. We have tested this hypothesis with aortic expiants from rats of the spontaneous autoimmune diabetic BB/Wor group, including diabetes-resistant, diabetes-prone, and treated acutely (<2 wk after onset of hyperglycemie) and chronically (>2 wk) diabetic strains. As a group, the VSMCs from BB/Wor animals showed enhanced outgrowth in 0.1% serum (23–48%) compared with VSMCs from ordinary Wistar rats (<10%). Within the BB/Wor group, however, the rank order of enhanced outgrowth was diabetes-resistant > chronically diabetic > acutely diabetic subjects. When the outgrowth assay was performed in the presence of supplemental insulin (10 mU/ml), outgrowth was increased, especially for the diabetes-prone animals, giving a new rank order of diabetes-prone > acutely diabetic > diabetes-resistant > chronically diabetic subjects. These data suggest a genetic predilection in the entire BB/Wor rat group for increased VSMC responsiveness to migratory and proliferative stimuli, a sensitivity of these VSMCs to insulin, and the dissociation of this vascular cell effect from other manifestations of diabetes, e.g., hyperglycemia.

TGF-β1 Induces an Accumulation of Connexin43 in a Lysosomal Compartment in Endothelial Cells

We have been studying the relationships between cell growth and the expression of the gap junction protein Connexin43 (Cx43) in cultured bovine aortic endothelial cells (BAEC). As part of these studies, we examined the effect of the growth inhibitory cytokine TGF-beta1 on Cx43 expression. We have shown recently that TGF-beta treatment increases Cx43 mRNA and synthesis, content, and half-life of the protein within 24 h, which leads, over the course of days, to an accumulation of Cx43 in large, intensely immunostaining vesicles, filling much of the perinuclear cytoplasmic space. In the current study, based on their distribution and markers, we identified these vesicles as lysosomes/autophagosomes. Cx43 immunostaining and staining with a fluorescent probe for acidic compartments are coincident, as retention of a fluorescent-labeled low-density lipoprotein occurs in a similar pattern and the same staining pattern can be detected in the treated cells using other markers for lysosomal compartments. TEM revealed prominent lysosomal figures with considerable heterogeneous material. After withdrawal of TGF-beta, the accumulated Cx43 was cleared only slowly, with some brightly immunoreactive cells remaining even after 72 h. The prolonged appearance (based on immunoreactivity in situ and in immunoblots) of intact vesicular Cx43 in the treated cells suggests decreased degradation, resulting from impaired lysosomal activity. These data not only emphasize the importance of the lysosome in connexin degradation, but also show that TGF-beta can cause an alteration in lysosomal functioning, with implications for cellular metabolism.

Cardiovascular Gap Junction Proteins: Molecular Characterization and Biochemical Regulation

Gap junctions are plasma membrane specializations containing channels which permit the intercellular exchange of ions and small molecules. Gap junction channels are of central importance in electrically excitable tissues such as myocardium where cell-to-cell passage of ions allows propagation of action potentials. Gap junctions are also present in many non-excitable cells (for example endothelial cells) where they may facilitate intercellular exchange of nutrients, metabolites, and signaling molecules as well as ions. The present review will focus on molecular biological and biochemical studies that have enhanced our understanding of the molecular composition of cardiac and vascular gap junction channels and the regulation of the subunit proteins that form them.

Endogenous reactive oxygen metabolites mediate sublethal endothelial cell dysfunction during reoxygenation

PURPOSE Endothelial cells (EC) secrete vasoactive eicosanoids, which maintain organ blood flow. Because EC are a major source of eicosanoids, we studied the effects of reoxygenation on EC prostacyclin production. METHODS Bovine aortic EC cultures were exposed to 2 hours of normoxia, then 1 hour of hypoxia (PO2 = 10 +/- 3.5 mm Hg), followed by 1.5 hours of reoxygenation in either normal medium or medium plus either superoxide dismutase (SOD, 300 units/ml), catalase (1200 units/ml), allopurinol (5.0 x 10(-4) mol/L), or dinitrophenol (10(-4) mol/L). RESULTS Prostacyclin production decreased to 40% (p < 0.05) of basal prostacyclin production after 1 hour of hypoxia. EC reoxygenated with control medium recovered to 48% of basal prostacyclin production. EC reoxygenated in SOD resulted in recovery (p < 0.05) to 154% of basal prostacyclin production after 60 minutes. Catalase treatment resulted in recovery to 105% (p < 0.05) of basal prostacyclin production within 30 minutes of reoxygenation. Allopurinol treatment resulted in 77% recovery (p < 0.05) of basal prostacyclin production only during 30 minutes of reoxygenation. Dinitrophenol treatment resulted in significant (> or = 85%, p < 0.05) sustained recovery of basal prostacyclin production at 30, 60, and 90 minutes of experimental reperfusion. CONCLUSIONS The hypoxia-induced decrease in EC prostacyclin does not recover during reoxygenation. Catalase/SOD allowed return to baseline prostacyclin production during reoxygenation, implicating reactive oxygen metabolites as mediators of decreased eicosanoid biosynthesis. Recovery of prostacyclin production after 60 minutes reoxygenation with dinitrophenol but not allopurinol suggests a mitochondrial origin of the oxygen metabolites responsible for decreased prostacyclin biosynthesis.