Neonatal Rat Smooth Muscle Cells Research Articles

Targeting Alanines in the Hydrophobic and Cross-Linking Domains of Native Elastin with Isotopic Enrichment and Solid-State NMR Spectroscopy

Details of elastin’s complex conformational and dynamic heterogeneity were acquired from one- and two-dimensional solid-state nuclear magnetic resonance (ssNMR) experiments on hydrated elastin that is isotopically enriched at its alanines. Elastin’s abundant alanines are useful probes of the structural and dynamical microenvironments in its cross-linking and hydrophobic domains. High isotopic enrichment of alanines in neonatal rat smooth muscle cells (NRSMC) elastin was obtained with the inhibition of alanine transaminase, combined with an excess of [U–13C]alanine in the culture media. Due to the fast, large-amplitude motions, R-TOBSY was utilized with selective homonuclear decoupling schemes to resolve 13C-Ala peaks and confirm assignments. A data-driven approach is applied, as the interpretation of chemical shifts is based on the distribution of conformation-dependent 13C-Ala chemical shifts in proteins in multiple databases. Alanine populations in elastin’s hydrophobic domains are primarily random coil...

Ultraviolet light-induced modification of crosslinked hyaluronan gels.

Hyaluronan (HA) gels (hylans) crosslinked with divinyl sulfone (DVS) are highly biocompatible and can be structurally modified to obtain desired mechanical properties that are attractive for their use as tissue-engineering scaffolds. However, unmodified hylan gels are not good substrates for cell attachment or infiltration, likely as a result of their smooth surface and the highly anionic nature of HA. This study investigated whether the cell-adhering characteristics of hylan gels could be enhanced by irradiation with ultraviolet (UV) light, with or without prior dehydration. The attachment and proliferation of neonatal rat smooth muscle cells atop these gels was compared with that on unmodified (control; C) or dehydrated (D) gels. UV-induced changes to gel structure and chemistry were characterized by confocal and electron microscopy, and fluorphore-assisted carbohydrate electrophoresis (FACE). Cell attachment was sparse on both unmodified (C) and dehydrated (D) gels. Significantly higher levels of cell attachment were observed on the surface of irradiated (UV) and dehydrated-irradiated (DUV) gels, likely because of texturing of the gel surface by UV light. In addition, dehydration of gels before UV irradiation created irregular pore-like structures through which cells appeared to migrate into the interior. FACE assays demonstrated that UV-irradiation alters the chemistry of HA, causing limited breakdown of HA chains and DVS crosslinks within gel and possibly creating new crosslinks that have not yet been identified. Because the hylan gels are altered structurally and chemically, binding of cells to the material is likely to be more permanent than possible by other approaches, such as coating of cell-adhesive matrix factors on the gel surface, described previously. The significance of this work is that we have developed a technique for the modification of DVS-crosslinked HA (hylans) to enhance their performance as a cellular scaffold for tissue-engineering applications.

Endogenous activation of c-myc expression and DNA synthesis in serum-starved neonatal rat smooth muscle cells

Abstract Earlier studies have shown that smooth muscle cells (SMC) from arteries of neonatal and adult rats differ markedly in their in vitro growth characteristics. Since some of these differences may be relevant to the proliferation of SMC in atherosclerotic plaques we examined the expression of three proto-oncogenes ( c-fos, c-jun, and c-myc) and an SMC-specific differentiation marker (α-actin) in cultured SMC. In presence of serum cultured adult SMC contained lower levels of α-actin mRNA than neonatal cells. In neonatal cells serum-starvation resulted in a distinct increase in both c-myc and α-actin mRNA levels, whereas the expression of these genes appeared to be unaffected in adult cells. Transfer of adult SMC proliferating in the presence of fetal calf serum to serum-free medium for 48 h almost completely inhibited DNA synthesis, whereas transfer of neonatal SMC to serum-free medium reduced DNA synthesis only to about 50%. Serum-starved adult and neonatal SMC did not contain c-fos or c-jun transcripts, but in both cell types serum-stimulation resulted in a comparable increase in the expression of both genes. The present results demonstrate clear differences in the mechanisms regulating gene expression in adult and neonatal SMC.

The inhibitory effect of neonatal rat smooth muscle cells and elastic extracellular matrix on development of the newly seeded cell population in culture

Neonatal smooth muscle cells were seeded in standard plastic Falcon flasks, on top of another 2-month-old culture of the same cell population or on top of an acellular matrix prepared by removal of these cells. The effect of both complete and acellular layers on the production of elastin, collagen and total extracellular matrix (EM) proteins as well as on cell division was measured. Compared with the standard population grown on plastic, the complete cell layer almost completely prevented the newly seeded cells from dividing. The acellular matrix did not affect cell doubling but caused a distinct decrease in the production of EM components.

Processing of soluble elastin in cultured neonatal rat smooth muscle cells.

The synthesis and extracellular deposition of elastin by cultured neonatal rat aorta smooth muscle cells has been followed. The addition of beta-aminopropionitrile to the culture medium promotes accumulation of soluble precursors of elastin. Under such conditions, a protein possessing characteristics of a soluble elastin precursor with an apparent molecular weight of 77,000 was detected and partially purified. Pulse-chase studies suggested that this 77-kDa protein undergoes an extracellular, enzymatically catalyzed process to a 71-kDa protein. This 71-kDa protein is strikingly similar to tropoelastins isolated from other tissue systems, in which no evidence for higher molecular weight soluble precursors is at present available. Data presented in this communication suggest that the 77-kDa protein, which we have designated protropoelastin, represents a precursor to the tropoelastin moiety produced in the neonatal rat smooth muscle cell culture.