Mathematical modeling of the impact of actin and keratin filaments on keratinocyte cell spreading.

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.

Cloning and functional analysis of the swine eNOS promoter.

We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5' flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5' RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5' deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from -227 to -82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from -227 to -1290, while negative cis-regulatory elements may be present from -1290 to -1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at -68/-59 was involved in the formation of these complexes.

The effect of phenotype on mechanical stretch-induced vascular smooth muscle cell apoptosis.

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD.

Developmental expression of endothelin receptors in postnatal swine mesenteric artery.

Studies were conducted to determine whether endothelin (ET) ETA and ETB receptor protein and mRNA expression is developmentally regulated in the postnatal swine mesenteric circulation. To this end, Western blotting and real-time reverse PCR were performed on protein and total RNA isolated from the mesenteric artery harvested from 3-, 10-, and 30-d-old swine. Western blot analysis revealed that ETA and ETB receptor protein expression in the swine mesenteric artery decreased over the age range studied; thus, ETA and ETB receptor protein expression was significantly greater in the 3-d-old group then progressively declined over the first postnatal month. Similar to the Western data, real-time PCR analysis revealed that ETA and ETB receptor mRNA expression also decreased over the age range studied; thus, ETA and ETB receptor mRNA expression was significantly greater in the 3-d-old group then progressively declined over the first postnatal month. Immunohistochemistry localized the ETA receptor to the vascular smooth muscle and the ETB receptor to the endothelial cell layer. Additionally, we report a partial cDNA sequence for the swine ETB receptor. We conclude that ETA and ETB receptor protein and mRNA expression is developmentally regulated in the postnatal swine mesenteric artery, being expressed to a greater degree in younger animals.

Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells.

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C >> alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.

Development of the myogenic response in postnatal intestine: role of PKC.

Previous attempts to determine developmental changes in the vascular myogenic response have been confounded by the presence of competing vasoactive stimuli or the use of isolated vessels with markedly different baseline diameters. To circumvent these issues, small mesenteric arteries (diameter approximately 150 microm) from 1- and 10-day-old piglets were studied in vitro under no-flow conditions. In situ studies demonstrated that the intravascular pressure and diameter of these vessels were similar in both age groups, allowing an effective comparison of the myogenic response not obscured by differences in basal diameter. The pressure-diameter relationship was age specific. Thus, although small mesenteric arteries from both age groups demonstrated myogenic constriction in response to stepwise increases in pressure (0 to 100 mmHg, in 20-mmHg increments), the intensity of contraction was significantly greater in vessels from 1-day-old piglets particularly within the pressure range normally experienced by these vessels in situ. Attenuation or activation of PKC with calphostin C or indolactam, respectively, substantially altered the pressure-diameter relationship in 1-, but not 10-day-old arteries; thus calphostin C essentially eliminated the contractile response to pressure elevation in younger subjects, whereas indolactam significantly increased the intensity of the myogenic response and shifted its activation point to a lower pressure range. Immunoblots carried out on protein recovered from these arteries revealed the presence of alpha, beta, epsilon, iota, and lambda; notably, expression of the alpha- and epsilon-isoforms substantially decreased between postnatal days 1 and 10.

Developmental expression of eNOS in postnatal swine mesenteric artery.

Developmental changes in the expression of endothelial nitric oxide synthase (eNOS) within the mesenteric artery of swine were studied in fetal (110 days postconception/117 days total gestation) and on postnatal days 1, 3, 10, and 30. Subjects in the 1-day-old group were subdivided into fed and nonfed. Transcription of eNOS was determined by real-time PCR, protein expression was evaluated by Western blotting, and hemodynamic and oxygenation parameters were measured within in situ gut loops before and after the administration of N(G)-monomethyl-L-arginine (L-NMMA). The abundance of eNOS mRNA remained steady throughout all ages. In contrast, expression of eNOS protein was twofold greater in the 1-day-old fed subjects compared with fetal or 1-day-old nonfed subjects. eNOS protein expression remained elevated on day 3, increased on day 10, and then declined to a level similar to the day 1 nonfed group by postnatal day 30. Intestinal vascular resistance was 31% lower in the day 1 fed group when compared to the day 1 nonfed group; resistance continued to decline through day 10 but then significantly increased on day 30. We conclude that the expression of eNOS changes within the mesenteric artery during early postnatal development at a posttranscriptional level.

Activated monocytes induce smooth muscle cell death: role of macrophage colony-stimulating factor and cell contact.

BACKGROUND: Plaque disruption is the inciting event for coronary thrombosis and acute coronary syndromes. Multiple factors influence plaque rupture, including the loss of vascular smooth muscle cells (VSMCs). We hypothesized that monocytes/macrophages (MMs) activated by macrophage colony-stimulating factor (M-CSF) are responsible for VSMC death. METHODS AND RESULTS: VSMC apoptosis was markedly increased in the presence of both M-CSF and MMs (58.8+/-3.3%) compared with VSMCs plus M-CSF without MMs (15.7+/-1.5%, P< or =0.00005), VSMCs plus MMs without M-CSF (22.7+/-3.7%, P< or =0.0001), or control VSMCs alone (13.2+/-2.1%, P< or =0.0001). MM cell contact was required for M-CSF-stimulated killing of VSMCs, and MMs displayed an M-CSF concentration-dependent killing effect. Abciximab binds Mac-1 (CD11b/CD18) on MMs. When added to VSMCs exposed to MMs and M-CSF, abciximab (7 microg/mL) significantly reduced VSMC apoptosis (19.1+/-2.2%, P< or =0.0003). Therapeutic doses of tirofiban (0.35 microg/mL) and eptifibatide (5 microg/mL), which inhibit platelet glycoprotein (GP) IIb/IIIa but not Mac-1, did not block activated MM-induced VSMC apoptosis (65.0+/-3.4% and 51.3+/-2.5%, respectively). A recombinant anti-CD-18 antibody had an effect similar to that of abciximab (16.5+/-0.4%). CONCLUSIONS: These data suggest that monocytes and physiological concentrations of M-CSF trigger VSMC apoptosis. Abciximab and specific inhibitors of the Mac-1 receptor can antagonize this process.