PubMed Health⌕ Search

Biomedical subjects

I Mills

Publications and source records attributed to I Mills.

At least 19 recordsLinked to original sources
PubMed1999

Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain.

The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

Animals↗
PubMed1998

Antiproliferative effect of elevated glucose in human microvascular endothelial cells.

Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1-14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10-50 microM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular pathophysiology associated with diabetic microangiopathy.

Cell Division↗
PubMed1998

Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain.

We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain.

Animals↗
PubMed1998

Induction of interleukin (IL)-1 alpha and beta gene expression in human keratinocytes exposed to repetitive strain: their role in strain-induced keratinocyte proliferation and morphological change.

Recent studies in our laboratory have demonstrated that mechanical strain alters many facets of keratinocyte biology including proliferation, protein synthesis, and morphology. IL-1 is known to play an important role in the autocrine regulation of these basic cellular properties under basal and stimulated conditions. However, it is not known whether IL-1 plays a role in strain-induced alteration of keratinocyte biology. Thus, the objective of this study was to test the hypothesis that cyclic strain stimulates IL-1 expression and that strain-induced changes in keratinocyte function is regulated by IL-1. To test this hypothesis, we examined the effect of cyclic strain (10% average deformation) on keratinocyte IL-1 gene expression and the effect of neutralizing antibodies of IL-1 alpha and IL-1 beta on strain-induced changes in keratinocyte proliferation, morphology, and orientation. Northern blot analyses demonstrated that steady state levels of IL-1 alpha and beta mRNA were elevated by 4 h, peaked at 1 2 h of cyclic strain (IL-1 alpha, 304+/-14.2%; IL-1 beta, 212+/-5.6% increase vs. static controls) and decreased gradually by 24 h. IL-1 antibodies (IL-1 alpha, 0.01 microg/ml; IL-1 beta, 0.01 microg/ml) significantly blocked strain-induced keratinocyte proliferation as well as the basal rate of proliferation. In contrast, IL-1 antibodies (IL-1 alpha, 0.01 microg/ml; IL-1 beta, 0.1 microg/ml) had no effect on strain-induced morphological changes such as elongation and alignment. We conclude that mechanical strain induces IL-1 mRNA expression in keratinocytes. The role of IL-1 in mediating strain-induced changes in keratinocyte biology remains to be determined but appears to be independent of morphological changes.

Antibodies↗
PubMed1998

Molecular basis for tissue expansion: clinical implications for the surgeon.

A wide variety of tissue expansion techniques have been used for breast reconstruction, craniofacial surgery, and burn care in plastic reconstructive surgery. However, the basic mechanism by which skin and surrounding tissue respond to mechanical expansion remains unclear. Recent studies have revealed the biomechanical aspects of cells subjected to strain and various factors involved in the stretch-induced signal transduction pathway. In this regard, we have reported previously that mechanical force increases keratinocyte growth and protein synthesis and alters cell morphology. The mechanism by which strain causes an enhancement of cellular growth appears to be a network of several integrated cascades, implicating growth factors, cytoskeleton, and the protein kinase family. Recently, additional evidence has accumulated that mechanical strain stimulates signal transduction pathways that could trigger a series of cascades eventually leading to a new skin production. For example, we have evidence suggesting a key role for protein kinase C (PKC) in mechanosignaling as PKC is activated and translocated in keratinocytes subjected to strain in an isoform-specific manner. In this report, molecular mechanisms leading to enhancement of skin surface area are reviewed, and possible future applications are discussed.

Burns↗
PubMed1997

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes.

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 +/- 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC alpha and delta, but not zeta isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC beta was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC.

Biological Transport↗
PubMed1997

Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain.

The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

Adenylyl Cyclases↗
PubMed1997

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways.

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

Activating Transcription Factor 2↗
PubMed1997

Effect of strain on human keratinocytes in vitro.

Tissue expansion, a technique to enlarge the skin surface area with an expandable balloon, has been widely used in reconstructive surgery. Although the effect of tissue expansion on in vivo skin physiology and histology has been well documented, it remains unclear whether keratinocytes or other cell types are responsible for these changes. Therefore, we investigated the in vitro effect of cyclic (10 cycles/min, 150 mmHg) or constant (continuous, 150 mmHg) strain on human keratinocyte phenotype and relevant mechanosignaling pathways. Our results demonstrate that keratinocytes subjected to cyclic strain exhibit a significant (P < 0.05) increase in cell proliferation (49.2+/-15.8%), DNA synthesis (37.7+/-4.5%), elongation (20.3+/-2.7%), and protein synthesis (17.9+/-6.6% increase) as compared with stationary controls. In contrast, keratinocytes subjected to constant strain were unaffected aside from a modest transitory increase in the proliferative rate. Keratinocytes subjected to cyclic strain aligned perpendicular to the force vector (24.2+/-1.6 degrees) as compared with stationary controls (40.4+/-2.2 degrees; the smaller degree indicates better alignment). We also report strain-induced reduction in the levels of cyclic adenosine mono phosphate (cAMP), protein kinase A (PKA), and prostaglandin E2 (PGE2) as compared with stationary controls (cAMP, 30+/-7.5%; PKA, 45+/-17%; PGE2, 58+/-4.3%; percent decrease vs. that of control). We conclude that direct application of cyclic strain on human keratinocytes modulates cell phenotype and cAMP-mediated signaling pathways in an inverse manner. Moreover, keratinocytes may play an important role in previously observed alterations in skin properties associated with tissue expansion and other strain-induced responses.

Cell Division↗
PubMed1997

Cyclic strain is a weak inducer of prostacyclin synthase expression in bovine aortic endothelial cells.

Recent studies indicate that hemodynamic forces such as cyclic strain and shear stress can increase prostacyclin (PGI2) secretion by endothelial cells (EC) but the effect of these forces on prostacyclin synthase (PGIS) gene expression remains unclear and is the focus of this study. Bovine aortic EC were seeded onto type I collagen coated flexible membranes and grown to confluence. The membranes and attached EC were subjected to 10% average strain at 60 cpm (0.5 sec deformation alternating with 0.5 sec relaxation) for up to 5 days. PGIS gene expression was determined by Northern blot analysis and protein level by Western blot analysis. The effect of cyclic strain on the PGIS promoter was determined by the transfection of a 1-kb human PGIS gene promoter construct coupled to a luciferase reporter gene into EC, followed by determination of luciferase activity. PGIS gene expression increased 1.7-fold in EC subjected to cyclic strain for 24 hr. Likewise, EC transfected with a pGL3B-PGIS (-1070/-10) construct showed an approximate 1.3-fold elevation in luciferase activity in EC subjected to cyclic strain for 3, 4, 8, and 12 hr. The weak stimulation of PGIS gene expression by cyclic strain was reflected in an inability to detect alterations in PGIS protein levels in EC subjected to cyclic strain for as long as 5 days. These data suggest that strain-induced stimulation of PGIS gene expression plays only a minor role in the ability of cyclic strain to stimulate PGI2 release in EC. These findings coupled with our earlier demonstration of a requisite addition of exogenous arachidonate in order to observe strain-induced PGI2 release, implicates a mechanism that more likely involves strain-induced stimulation of PGIS activity.

Animals↗
PubMed1997

Recruiting general practitioners to rural areas: one community's experience.

The rural community of Dalwallinu found itself without a medical practitioner early in 1995. Believing recruitment of a replacement practitioner would be relatively straightforward, the Shire and the Hospital Board combined for this process. Little did we realise that there would be many factors that would impinge upon our ability to recruit a general practitioner (GP) other than the availability of a suitably qualified practitioner. Locum availability, providing a furnished house, a motor car, becoming the employer of the locum practitioner, community reactions, government regulations on recruiting doctors trained overseas, Health Department restructures and the arbitrary regulations of professions all had a significant impact, not to mention the cost to the local council to provide a community service.

Family Practice↗
PubMed1997

Gene regulation by mechanical forces.

Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

Animals↗
PubMed1996

Protein phosphatase 2A in stretch-induced endothelial cell proliferation.

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

Animals↗
PubMed1995

Cyclic strain causes heterogeneous induction of transcription factors, AP-1, CRE binding protein and NF-kB, in endothelial cells: species and vascular bed diversity.

Recent studies demonstrate that cyclic strain stimulates protein kinase C in bovine aortic endothelial cells (BAEC) as well as the induction of immediate early genes and the transcription factor activator protein-1 (AP-1) in human umbilical vein endothelial cells (HUVEC). The objective of this study was to determine whether transcriptional factor induction in endothelial cells (EC) exposed to strain is the same with regard to the species and vascular bed they are derived from. Evidence for a heterogeneous response for growth, orientation and prostacyclin secretion has been obtained for a variety of EC exposed to cyclic strain. In this study, we investigated cyclic strain mediated induction of transcription factors, AP-1, cAMP response element binding protein (CRE) and nuclear factor kB (NF-kB) in cultured EC from HUVEC, human aorta (HAEC), and BAEC. EC were exposed to 10% average strain at 60 cpm for up to 24 h. At varying time points, nuclear protein was extracted and analyzed for production of AP-1, CRE and NF-kB by electromobility shift assay. The results demonstrate that EC exposure to cyclic strain leads to a significant induction of AP-1, CRE and NF-kB in HAEC and HUVEC, but not in BAEC. Furthermore, these findings are in marked contrast to the previously described shear stress induced activation of AP-1 and NF-kB in BAEC. There was also a temporal difference in their response such that stretch-induced activation of AP-1 and NF-kB peaked at 4 h, whereas CRE increased in a biphasic manner at 15 min and 24 h. These results may partially explain the divergent effects of cyclic strain on EC gene expression and phenotype in EC from different vascular beds and species and underscore the difference in EC response to cyclic strain and shear stress.

Activating Transcription Factor 2↗
PubMed1994

Cyclic strain stimulates dephosphorylation of the 20kDa regulatory myosin light chain in vascular smooth muscle cells.

The role of cyclic strain in the regulation of 20 kDa myosin light chain phosphorylation (MLC20) in cultured smooth muscle cells (SMC) is unknown. The objective of this study was to determine whether cyclic strain stimulates the dephosphorylation of MLC20 in serum-fed SMC displaying a high basal level of phosphorylation. Confluent bovine aortic SMC were subjected to 10% average strain at 60 cycles per minute for 30 and 60 minutes. Basal MLC20 phosphorylation (N = non,M = mono,D = di) of serum-fed SMC was as follows: N = 34%:M = 27%:D = 39%. After 60 min of cyclic strain, both mono and diphosphorylated MLC20 were decreased to 21 and 15% respectively. The strain-induced dephosphorylation of MLC20 was partially inhibited by the protein phosphatase 1/2A inhibitor, calyculin A (5 nM). However, phosphorylase a phosphatase activities in Triton-soluble and insoluble fractions of SMC were unaffected by cyclic strain. The data suggest that cyclic strain causes dephosphorylation of MLC20 in SMC which may be partially due to activation of MLC20 phosphatase and/or inhibition of MLC20 phosphorylation.

Animals↗
PubMed1994

Chronic cyclic strain reduces adenylate cyclase activity and stimulatory G protein subunit levels in coronary smooth muscle cells.

Previous studies from this laboratory have demonstrated that acute cyclic strain causes a reduction in adenylate cyclase activity in cultured coronary vascular smooth muscle cells. The objective of this study was to test the hypothesis that chronic cyclic strain of coronary vascular smooth muscle cells also causes inhibition of adenylate cyclase activity and that this may be related to changes in G protein steady-state levels. Cultured smooth muscle cells obtained from porcine coronary artery were subjected to 24 h of cyclic strain of 20 kPa (24% maximum strain) at 60 cycles/min. Unstretched cells served as controls. Basal, Gpp(NH)p, and forskolin plus Mn(2+)-stimulated adenylate cyclase activities were inhibited significantly in stretched versus unstretched vascular smooth muscle cells. The reduction in adenylate cyclase activity observed after 24 h of cyclic strain was associated with a significant (P < 0.05 vs controls) reduction in steady-state levels of Gs alpha 45, whereas Gi alpha 1,2 and G beta levels remained unchanged. The data support the hypothesis that adenylate cyclase activity and G protein steady-state levels in coronary smooth muscle are sensitive to chronic cyclic strain. It suggests that the G protein adenylate cyclase effector pathway may play an important role in the subacute adaptation of the coronary circulation to changes in intravascular pressure.

Adenylyl Cyclases↗
PubMed1994

Induced mood, phobic responding and the return of fear.

The link between depression and phobic responding is poorly understood. Forty-eight spider phobics were exposed to videotaped spider sequences whilst in relatively depressed or relatively elated moods, then presented the same stimuli whilst in a neutral mood. Results indicated that depressed mood increased initial responding, and was associated with greater return of fear during the second (non-mood) sequence. The return of fear was not attributable solely to differential habituation rates during the first sequence, suggesting that the differences observed may have been due to cognitive effects.

Adolescent↗