Thomas H. Rosenquist

Homocysteine signal cascade: production of phospholipids, activation of protein kinase C, and the induction of c-fos and c-myb in smooth muscle cells.

Hyperhomocysteinemia has been recognized as an independent risk factor for cerebral, coronary, and peripheral atherosclerosis. To examine the contribution of homocysteine (H[cys]) in the pathogenesis of vascular diseases, we sought to determine whether the H[cys] effect on vascular smooth muscle (VSMC) proliferation is mediated by a specific receptor/transporter or is due to an interaction with growth factors or cytokines. We show that H[cys] induced c‐fos and c‐myb and increased DNA synthesis and cell proliferation 12‐fold in neural crest‐derived VSMC (N‐VSMC). The H[cys] effect on N‐VSMC proliferation is inhibited by Mk‐801, a noncompetitive antagonist of the N‐methyl‐D‐aspartate (NMDA) receptor, a glutamate‐gated calcium ion channel receptor, and CGS 19755, a competitive antagonist of NMDA‐type glutamate receptor. H[cys] stimulates the synthesis of mass amounts of sn‐1,2 diacylglycerol, and activates protein kinase C translocation from the nucleus and cytoplasm to cell membranes. Furthermore, protein kinase C inhibitors block the growth effect mediated by H[cys]. These findings indicate that H[cys]‐mediated responses are coupled to diacylglycerol‐dependent protein kinase C activation. Our results suggest that homocysteine activates a receptor/transporter‐like factor in neural crest derived smooth muscle.—Dalton, M. L., Gadson, P. F., Jr., Wrenn, R. W., Rosenquist, T. H. Homocysteine signal cascade: production of phospholipids, activation of protein kinase C, and the induction of c‐fos and c‐myb in smooth muscle cells. FASEB J. 11, 703–711 (1997)

Dextromethorphan and other N-methyl-D-aspartate receptor antagonists are teratogenic in the avian embryo model.

N-Methyl-D-aspartate (NMDA) receptors are a calcium-conducting class of excitatory amino acid receptors that are involved in neuronal development and migration. Certain well known teratogens (e.g. homocysteine, ethanol, and chloroform) that induce congenital neural tube and neural crest defects also have the capacity to act as NMDA receptor antagonists. We hypothesized that teratogenicity was a general property of NMDA receptor antagonists, and that high affinity NMDA receptor antagonists would induce neural tube and neural crest defects. Chicken embryos were given 5, 50, or 500 nmol/d of selected NMDA receptor antagonists for 3 consecutive days during the process of neural tube closure, beginning 4 h after the beginning of incubation. Selected NMDA receptor antagonists represented three classes of antagonists: ion channel blockers, glycine site antagonists, and glutamate site agonists and antagonists. All classes of NMDA receptor antagonists induced embryonic death and congenital defects of the neural crest and neural tube; however, the channel blockers were the most potent teratogens. Dextromethorphan at 500 nmol/embryo/d killed more than half the embryos and induced congenital defects in about one-eighth of the survivors; dextromethorphan was also highly lethal at 50 nmol/embryo/d. Glutamate site NMDA receptor agonists (NMDA and homoquinolinic acid) displayed weak toxicity relative to their known NMDA receptor potency. Taken together, these data indicate that NMDA receptor antagonists, particularly channel blockers, are potent teratogens in the chicken embryo model. Because dextromethorphan is a widely used nonprescription antitussive, its strong teratogeneticity using this model is particularly noteworthy.

Importance of folate-homocysteine homeostasis during early embryonic development

Abstract Although the beneficial effects of maternal folate supplementation in the periconceptional period have been shown to prevent neural tube defects, congenital heart defects and orofacial clefts, the exact protective mechanism of folates remains unknown. Folates affect DNA synthesis, amino acid metabolism and methylation of genes, proteins and lipids via S-adenosylmethionine-mediated one-carbon transfer reactions. Our laboratory has created several mouse knock out models of folate transport using gene targeting to inactivate folate receptor 1 (Folr1), folate receptor 2 (Folr2) and reduced folate carrier 1 (Slc19a1) genes. Gene ablation of both Folr1 and Slc19a1 leads to lethality, but with maternal folate supplementation, nullizygous embryos for both genes present with neural tube defects (NTDs) and congenital heart defects (CHDs). Folr1 nullizygous mice also exhibit orofacial clefts when the dams are provided with low folate supplementation during pregnancy. Finally, women with NTD-affected pregnancies have been reported to have high autoantibody titers against the folate receptor, potentially inhibiting the transport of folate to the developing embryo. This may be an explanation for some of the folate-responsive NTDs and perhaps other congenital malformations. Herein, we propose how homocysteinylation of the folate receptor may contribute to generation of these autoantibodies against the folate receptor. Clin Chem Lab Med 2007;45:1717–27.

N-methyl-D-aspartate receptor agonists modulate homocysteine-induced developmental abnormalities

We showed previously that the induction of neural crest (NC) and neural tube (NT) defects is a general property of N‐methyl‐D‐aspartate receptor (NMDAR) antagonists. Since homocysteine induces NC and NT defects and can also act as an NMDAR antagonist, we hypothesized that the mechanism of homocysteine‐induced developmental defects is mediated by competitive inhibition of the NMDAR by homocysteine. If this hypothesis is correct, homocysteine‐induced defects will be reduced by NMDAR agonists. To test the hypothesis, we treated chicken embryos during the process of neural tube closure with sufficient homocysteine thiolactone to induce NC and NT defects in ∼40% of survivors or with homocysteine thiolactone in combination with each of a selected set of NMDAR agonists in 0.05–5000 nmol doses. Glutamate site agonists selected were L‐glutamate and N‐methyl‐D‐aspartate. Glycine site agonists were glycine, D‐cycloserine, and aminocyclopropane‐carboxylic acid. Glycine was the most effective overall, reducing defects significantly at two different doses (each P<0.001). These results support the hypothesis that homocysteine may affect NC and NT development by its ability to inhibit the NMDAR. One potentially important consequence of this putative mechanism is that homocysteine may interact synergistically with other NMDAR antagonists to enhance its effect on development.—Rosenquist, T. H., Schneider, A. M., and Monaghan, D. T. N‐methyl‐D‐aspartate receptor agonists modulate homocysteine‐induced developmental abnormalities. FASEB J. 13, 1523–1531 (1999)

Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation

BACKGROUND The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation. METHODS RFC1(+/-) males were mated to RFC1(+/-) females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice. RESULTS Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5-E10.5, but they are developmentally delayed relative to wild-type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin. CONCLUSIONS High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin.

Connections between stereocilia in auditory hair cells of the alligator lizard

The interconnections between stereocilia within individual tufts of auditory hair cells in the basilar papilla of the alligator lizard were examined with a transmission electron microscope. An elaborate array of fibers near the base of each stereocilium (where it tapers to anchor into the cuticular plate) connected it to each of its neighboring stereocilia. The tips of individual stereocilia, which were slightly larger in diameter than their shaft, contacted adjacent stereocilia. Fibers also connected the tip of the kinocilium to neighboring stereocilia in the first row. The remaining regions of the stereocilia were relatively free of connecting fibers. The integrity of these connecting fibers are likely to be important in maintaining the normal micromechanical tuning and mechanoelectric transduction in these auditory hair cells. The addition of 0.1% ruthenium red to the primary fixative enhanced the preservation of the connecting structures, implying the presence of glycosaminoglycans.

The Pearson Silver-Gelatin Method for Light Microscopy of 0.5-2 μ Plastic Sections

Sections 0.5-2 μ thick of liver, kidney, lung, cartilage and brown fat embedded in Maraglas, Araldrite, Epon and Spurrs medium were deplasticized in alcoholic NaOH for 15 min. Following several alcohol rinses, the tissues were exposed to 2% AgNO3 at 50 C for 1-2 hr, and then developed in a solution containing 3% gelatin, 40 ml; 2% AgNO3, 10 ml; and 1% hydroquinone, 4 ml. Sections were then toned in 1% gold chloride (several dips), washed in water for 5 min and dipped in 2% oxalic acid. After a brief rinse in water the sections were placed in 5% Na2S2O3 for 5 min, washed in water for 5 min, dehydrated in alcohol, cleared and covered. Compared to similar sections retained in plastic and deplasticized sections stained in the routine manner with 1% toluidine blue, silver impregnated sections of all tissue free of plastic, displayed greater clarity of cellular detail. Especially clear were mitochondria of liver and brown fat cells. Type II cells of the lungs were exceptionally prominent as were podocyte foot ...

Transforming growth factor-beta: Signal transduction via protein kinase C in cultured embryonic vascular smooth muscle cells

SummaryTransforming growth factor-beta (TGF-β), an ubiquitous regulatory peptide, has diverse effects on the differentiation and behavior of vascular smooth muscle cells (VSMC). However, the molecular mechanism through which TGF-α exerts its effects remains obscure. We investigated the phosphoinositide/protein kinase C [PKC] signaling pathway in the action of TGF-β on cultured embryonic avian VSMC of differing lineage: a) thoracic aorta, derived from the neural crest; and b) abdominal aorta, derived from mesenchyme. The second messenger responsible for activation of PKC is sn-1,2-diacylglycerol [DAG]; TGF-β increased the mass amounts of DAG in the membranes of neural crest-derived VSMC concurrent with translocation of PKC from the soluble to the membrane fraction, but TGF-β had no effect on the DAG or PKC of mesenchyme-derived VSMC. TGF-β potentiated the growth of platelet-derived growth factor (PDGF)-treated, neural crest-derived VSMC; but abolished PDGF-induced growth of mesenchymal cells. It is concluded that molecular and functional responses of VSMC to TGF-β are heterogeneous and are functions of the embryonic lineage of the VSMC.

Expression of elastin, smooth muscle alpha-actin, and c-Jun as a function of the embryonic lineage of vascular smooth muscle cells

SummaryIn the avian embryo, vascular smooth muscle cells (VSMC) in the aortic arch (elastic) arteries originate in the neural crest, whereas other VSMC develop from local mesoderm. These two lineages have been shown previously to be significantly different in the timing and expression of the smooth muscle phenotype and in their respective abilities to produce an orderly elastic matrix. Two differing kinds of VSMC also have been shown in mammals. In the experimental absence of neural crest (NC) in the avian embryo, the matrix is spatially disordered. The molecular basis of the difference between the normal NC-VSMC and the surrogate mesodermal (MDM)-VSMC has not previously been investigated. In this study the expression of vascular smooth muscle alpha-actin, tropoelastin, c-fos and c-jun were examined via immunoblotting, immunohistochemistry, Northern blot, and/or transcription run-on assays. Control avian VSMC of NC origin were compared with experimental MDM-derived VSMC that populate the cardiac outflow after surgical ablation of the NC. The results show that, when they are grown under identical conditions in vitro or freshly removed from an embryonic vessel, surrogate MDM-VSMC express about 10 times more alpha-actin and tropoelastin than the normal NC-VSMC; and MDM-VSMC express up to 15 times more c-jun, whereas c-fos was not different. These results show profound heterogeneity in the regulation of VSMC-specific genes that is based in the embryonic lineage of the cells.

The structure of the pulmonary intervalveolar microvascular sheet

Abstract To understand the morphologic basis of the compliance of the pulmonary interalveolar microvascular sheet, we investigated the histology and ultrastructure of the adult beagle interalveolar septum with special emphasis on the “posts” of the intercapillary interstitium. Special stains for collagen and elastin for light and electron microscopy were used; control preparations included tissues exposed to elastase and hyaluronidase prior to staining. In the interalveolar wall remote from vessels and ducts fine collagen fibers surround the posts, corss over the posts to adjacent capillaries, and pass through the posts from one side of the septum to the other in a curving arrangement. In cross-section the overall post configuration is biconcave and collagen is found at post borders in its course through the post. Elastin is more abundant and coarser than collagen, passes linearly through the posts, and is always intimately associated with collagen including the region of the alveolar-capillary membrane in the respiratory area. Following elastase hydrolysis, non-collageneous elements do not stain. Removal of post ground substance with hyaluronidase disrupts the fibroprotein pattern of connective tissue.