Charles L. Seidel

Differentiated vascular myocytes: are they involved in neointimal formation?

The role of differentiated vascular myocytes are neointimal formation in canine carotid artery was investigated. Using antibodies and cDNA probes, cells were characterized in situ and after isolation. In situ characterization indicated the majority of medial cells expressed both smooth muscle myosin and alpha actin but many cells were negative to these markers. All adventitial cells were negative for these proteins. The muscle protein-positive cells were designated differentiated, vascular myocytes (VSMC). The others were designated type 2 cells. Sequential enzyme digestion from lumenal surface yielded VSMC ( > 90%) while digestions from the adventitial surface yielded type 2 cells ( > 90%). VSMC were viable in culture but did not spread, proliferate, or alter expression of muscle proteins. Type 2 cells proliferated and increased their expression of muscle actin but did not express muscle myosin. Characterization of neointimal cells from injured carotid arteries indicated they were morphologically and immunologically identical to cultured type 2 cells. We concluded that: (a) canine carotid artery media consists of a heterogeneous cell population: (b) serum does not stimulate isolated VSMC to undergo phenotypic modulation or proliferate: and (c) type 2 cells may be responsible for neointimal formation because they proliferate and acquire a phenotype identical to in situ neointimal cells.

Insulin reduces contraction and intracellular calcium concentration in vascular smooth muscle.

Resistance to insulin-induced glucose disposal is associated with hypertension, in accord with recent reports that insulin-induced vasodilation is impaired in men with resistance to insulin-induced glucose disposal. Nevertheless, the mechanism of insulin-induced vasodilation is not known. We wished to determine whether a physiological concentration of insulin inhibits agonist-induced contraction at the level of the individual vascular smooth muscle cell, and if so, how. Dispersed vascular smooth muscle cells from dog femoral artery were grown on collagen gels for 4 to 8 days. Contraction and intracellular Ca2+ concentration of individual cells were measured by photomicroscopy and fura 2 epifluorescence microscopy, respectively. Serotonin and angiotensin II contracted cells in a dose-dependent manner. Preincubation of cells for 20 minutes (short-term) or 7 days (long-term) with insulin (40 microU/mL) inhibited serotonin- and angiotensin II-induced contractions by approximately 50%. Insulin (10 microU/mL) acutely inhibited serotonin-induced contraction by 34%. The maximal effect of high extracellular K(+)-induced contraction was not affected by short-term insulin exposure, but the ED50 for extracellular K(+)-induced contraction was increased from 7.6 +/- 2.5 to 16.0 +/- 3.9 mmol/L (P < .05). Short-term insulin exposure also attenuated the peak rise of the serotonin-induced intracellular Ca2+ transient and increased the rate constant for intracellular Ca2+ decline. Verapamil and ouabain completely blocked the attenuation of agonist-induced contraction by short-term insulin exposure, indicating the importance of voltage-operated Ca2+ channels and the Na(+)-K+ pump for this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

A Comparison of In-Class Learner Engagement Across Lecture, Problem-Based Learning, and Team Learning Using the STROBE Classroom Observation Tool

Background: Having recently introduced team learning into the preclinical medical curriculum, evidence of the relative impact of this instructional method on in-class learner engagement was sought. Purpose: To compare patterns of engagement behaviors among learners in class sessions across 3 distinct instructional methods: lecture, problem-based learning (PBL), and team learning. Methods: Trained observers used the STROBE classroom observation tool to measure learner engagement in 7 lecture, 4 PBL, and 3 team learning classrooms over a 12-month period. Proportions of different types of engagement behaviors were compared using chi-square. Results: In PBL and team learning, the amount of learner-to-learner engagement was similar and much greater than in lecture, where most engagement was of the learner-to-instructor and self-engagement types. Also, learner-to-instructor engagement appeared greater in team learning than in PBL. Conclusions: Observed engagement behaviors confirm the potential of team learning to foster engagement similar to PBL, but with greater faculty input.

Team Learning in Medical Education: Initial Experiences at Ten Institutions

Purpose. In the midst of curricular reforms that frequently call for reducing lectures and increasing small-group teaching, there is a crisis in faculty time for teaching. This paper describes the initial experiences of ten institutions with team learning (TL), a teaching method which fosters small-group learning in a large-class setting. Method. After initial pilot studies at one institution, nine additional institutions implemented TL in one or more courses. Results. Within 18 months, TL has been used in 40 courses (from .5% to 100% of the time) and all ten institutions will increase its use next year. Conclusions. We surmise that this relatively rapid spread of TL into the medical curriculum is due to the sound pedagogy and efficiency of TL as well as the modest financial resources and support we have provided to partner institutions.

Cellular Heterogeneity of the Vascular Tunica Media Implications for Vessel Wall Repair

Following arterial injury in adult humans and other mammals, there occurs a thickening of the intimal layer of the vessel wall due to migration of cells from the tunica media and proliferation of these migrated cells, along with any resident intimal cells. This intimal thickening, or neointimal formation, can lead to vessel stenosis or even occlusion and is the cellular basis of all intimal vascular disease. An understanding of this process leading to prevention, retardation, or reversal of intimal thickening would dramatically reduce morbidity and mortality from vascular disease. The identity of the cells involved in intimal thickening has not been conclusively determined. The morphology, growth properties, and protein expression of cells in the thickened intima are distinct from those of vascular smooth muscle cells within the tunica media or of endothelial cells lining the vessel lumen. There are at least three hypotheses to explain the identity of neointimal cells: (1) They arise from fully differentiated vascular smooth muscle cells within the tunica media. During their migration to and proliferation in the intimal layer, they undergo function-specific modifications, thus acquiring the characteristics ascribed to neointimal cells. (2) They arise from a normally resident population of smooth muscle “stem cells.” Such cells may be embryonic or fetal smooth muscle cells that have not fully differentiated or a multipotential cell that could form either neointimal or smooth muscle cells. Unlike differentiated smooth muscle cells, these cells would retain the ability to migrate and proliferate. During migration to and subsequent proliferation within the thickening intima, such cells may acquire additional characteristics of neointimal cells. (3) They arise from a cell lineage within the tunica media that is distinct from that of the smooth muscle cell. The characteristics of these cells may be modulated to those of neointimal cells …

Validation Of An Observation Instrument For Measuring Student Engagement In Health Professions Settings

Documenting student engagement has received increased emphasis in medical schools, as teaching strategies are changing to include more student-to-student interactions. The purpose of this study was to develop and evaluate a measure of student engagement completed by independent observers that would not interfere with student learning time. Data from 3,182 observations completed by nine observers in 32 educational classroom settings with 23 different instructors were used to evaluate the interobserver reliability and gather validity evidence for our observational instrument, named the STROBE. Results indicated that interobserver agreement was good to excellent when observations were conducted simultaneously on randomly selected students in the same classroom (84% average agreement and 0.79 average kappa coefficient) and when observations were conducted on different randomly selected students (79% average agreement). Results also provided strong evidence for validity. Overall, findings indicate that the STROBE demonstrates promise for educational research and evaluation by documenting student engagement in medical education settings.

Insulin Acutely Inhibits Cultured Vascular Smooth Muscle Cell Contraction by a Nitric Oxide Synthase–Dependent Pathway

Insulin acutely decreases contractile agonist-induced Ca2+ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca2+ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase-dependent pathway. Primary cultures of endothelial cell-free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10(-5) mol/L serotonin insulin stimulated cGMP production by 60%. N(G)-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)-stimulated Mn+2 (a Ca2+ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT-stimulated Ca2+ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase-dependent mechanism.

Insulin-Stimulated Glucose Transport Inhibits Ca2+ Influx and Contraction in Vascular Smooth Muscle

BACKGROUND Insulin attenuates serotonin-induced Ca2+ influx, the intracellular Ca2+ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca2+ influx, intracellular Ca2+ concentration, and contraction in these cells. METHODS AND RESULTS Insulin 1 nmol/L stimulated the 30-minute uptake of [3H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca2+ concentration was monitored by measuring fura 2 fluorescence by use of Ca(2+)-sensitive excitation wavelengths, and Ca2+ influx was estimated by the rate of Mn2+ quenching of intracellular fura 2 fluorescence when excited at a Ca(2+)-insensitive wave-length. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10(-5) mol/L serotonin-induced contraction of individual cells by 62% (P < .01) and decreased the serotonin-stimulated component of Mn2+ influx by 78% (P < .05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca2+ peak by 37% (P < .05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn2+ influx, the serotonin-induced Ca2+ peak was decreased by 46% (P < .05), serotonin-induced contraction was inhibited by 60% (P < .01), and addition of insulin did not further inhibit contraction. CONCLUSIONS Since the effects of insulin on serotonin-stimulated Ca2+ transport, intracellular Ca2+ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca2+ influx and contraction in vascular smooth muscle.

Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase.

Vascular smooth muscle cell (VSMC) migration participates in atherosclerosis and arterial restenosis after balloon angioplasty. Because these processes are enhanced in insulin-resistant states, our goal was to determine whether insulin affects VSMC migration and, if so, how. The migration of primary cultured VSMCs from canine femoral artery was measured with the use of a wound migration assay and related to cGMP levels. Insulin (1 nmol/L) did not affect migration or cGMP production in control cells. When inducible nitric oxide synthase (iNOS) was induced by 24-hour preincubation with lipopolysaccharide and interleuken-1beta, basal migration decreased, cGMP production increased, and insulin inhibited migration by >90% and stimulated cGMP production by 3-fold. The nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine blocked the affect of insulin on the migration of VSMCs with iNOS. 8-Bromo-cGMP inhibited VSMC migration in control cells, and 1-H-1[1,2,4]oxadiazolo-[4, 3a]quinoxolin-1-one, a selective inhibitor of guanylate cyclase, blocked the inhibition by insulin of migration of cells with iNOS. We conclude that insulin does not normally affect cGMP production or the migration of these VSMCs. However, after the induction of iNOS, insulin stimulates cGMP production and inhibits migration via an NOS-and a cGMP-dependent mechanism.

Insulin inhibits serotonin-induced Ca2+ influx in vascular smooth muscle.

Insulin in physiological concentrations attenuates the agonist-induced intracellular Ca2+ ([Ca2+]i) transient and inhibits contraction in individual nonproliferated cultured canine femoral artery vascular smooth muscle cells (VSMCs). In the present study, we wished to define the effects of insulin on individual components of Ca2+ transport in vascular smooth muscle. Methods and ResultsInsulin (40 μU/mL) attenuated the 5-hydroxytryptamine (5-HT, serotonin; 10−5 mol/L)-induced [Ca2+]i transient (measured by fura 2 fluorescence) in primary confluent canine femoral artery VSMCs in the presence of extracellular Ca2+. In Ca2+ -free media, the 5-HT-induced [Ca2+ transient was reduced by 42% and was not affected by insulin. This finding suggested that insulin inhibits 5-HTinduced Ca2+ influx but does not affect sarcolemmal Call efflux or Ca2+ release from intracellular stores. In support of those conclusions, we found that insulin inhibited the 5-HT-induced component of Mn2+ (a Ca2+ surrogate) influx (measured by fura 2 fluorescence quenching at the Ca2+ isosbestic excitation wavelength). In addition, 5-HT stimulated the rates of 45Ca2+ efflux from intact cells (a measure of sarcolemmal Ca2+ efflux) and from saponin-permeabilized cells (a measure of Ca2+ release from intracellular stores), but insulin did not affect these rates of 45Ca2+ efflux. ConclusionsWe conclude that a physiological insulin concentration attenuates the 5-HT- induced [Ca2+]i transient in confluent primary cultured canine femoral artery VSMCs by inhibiting the 5 -HT-induced component of Ca2+ influx but not by affecting sarcolemmal Ca2+ efflux or Ca2+ release from intracellular stores.