Denise C. Polacek

Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function.

Abstract: Atherosclerosis originates at predictable focal and regional sites that are associated with complex flow disturbances and flow separations in large arteries. The spatial relationships associated with hemodynamic shear stress forces acting on the endothelial monolayer are considered in experiments that model regions susceptible to atherosclerosis (flow disturbance) and resistant to atherosclerosis (undisturbed flow). Flow disturbance in vitro induced differential expression at the single gene level as illustrated for the intercellular communication gene and protein, connexin 43. Transcription profiles of individual endothelial cells isolated from both disturbed and undisturbed flow regions exhibited more expression heterogeneity in disturbed than in undisturbed flow. We propose that within highly heterogeneous populations of endothelial cells located in disturbed flow regions, proatherosclerotic gene expression may occur within the range of expression profiles induced by the local hemodynamics. These may be sites of initiation of focal atherosclerosis. Mechanisms are proposed to account for heterogeneous endothelial responses to shear stress by reference to the decentralized model of endothelial mechano‐transduction. Length scales ranging from centimeters to nanometers are useful in describing regional, single cell, and intracellular mechanotransduction mechanisms.

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall. Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients. The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments. Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.

A spatial approach to transcriptional profiling: mechanotransduction and the focal origin of atherosclerosis

The initiation and progression of focal atherosclerotic lesions has long been known to be associated with regions of disturbed blood flow. Improved precision in experimental models of spatially defined flow has recently been combined with regional and single-cell gene-expression profiling to investigate the relationships linking haemodynamics to vessel-wall pathobiology.

Connexin43 gene expression in the rabbit arterial wall: effects of hypercholesterolemia, balloon injury and their combination.

The specialized functions of endothelium require intercellular communication between endothelial cells within the monolayer, and between endothelium and other cells present in the vessel wall. This is accomplished by a combination of paracrine soluble mediators and direct gap-junctional intercellular communication (GJIC) mediated by a family of connexin proteins. A prominent connexin expressed by vascular cells in vivo and in vitro is connexin 43 (Cx43). We have investigated the in vivo gene regulation of Cx43 in the context of vascular pathology, as a result of mechanical injury, hypercholesterolemia or both. The aortoiliac bifurcation in the rabbit was examined following three types of insult: (1) diet-induced hypercholesterolemia resulting in macrophage-rich fatty streak lesions, (2) mechanical, stretch-denudation injury resulting in intimal smooth muscle cell (SMC) proliferation and (3) mechanical injury superimposed on hypercholesterolemia resulting in a complex vascular lesion having characteristics of both interventions. The normal rabbit iliac artery expressed approximately equal levels of Cx43 mRNA in the medial SMC layers and in the endothelium. In hypercholesterolemia-induced atherosclerosis, Cx43 expression was most prominent in macrophage foam cells even though normocholesterolemic precursor monocytes did not express Cx43 mRNA. Antibodies directed specifically to Cx43 protein confirmed the expression of macrophage gap junction protein in these cells. Medial SMC in hypercholesterolemia exhibited less Cx43 than their normal counterparts in control animals. Mechanical injury in the absence of hypercholesterolemia resulted in intimal thickening in which Cx43 expression in the intimal SMC was equivalent to that in the subjacent medial SMC, both being approximately equivalent to normal uninjured rabbit medial SMC expression. Cell-specific expression of Cx43 in combined mechanical injury/hypercholesterolemia was similar to that observed in hypercholesterolemia alone: Cx43 upregulation in macrophages, while medial SMC were downregulated. Normo- and hypercholesterolemic alveolar macrophages of the lung and Kupffer cells of the liver did not exhibit induction of Cx43 mRNA, nor did macrophages isolated from peritoneal or bronchial lavage fluid of the same animals. This work extends our previous finding of Cx43 upregulation in human atherectomy tissue and demonstrates that atherosclerotic lesions in situ, in a controlled animal model of atherosclerosis, exhibit cell-specific changes in Cx43 gene expression. Changes in medial SMC migration, proliferation and phenotype, as well as enhanced interactions between adherent/infiltrating monocytes and endothelium may be related to modified GJIC pathways in the vessel wall.

Rapid fractionation of human high density apolipoproteins by high performance liquid chromatography

A simple and rapid fractionation procedure (30 min) has been developed for the isolation of the major apoproteins from human serum high density lipoproteins by molecular sieving in a high performance liquid chromatographic column. Apo A-I, apoA-II and the C peptides are quantitatively resolved up to a protein load of 3 mg. The technique has also been successfully applied to the final purification of A apoproteins which has been isolated by conventional chromatographic procedures and as a sensitive analytical tool for assessing apoprotein purity.

Changes in gap junction connexin-43 messenger ribonucleic acid levels associated with rat ovarian follicular development as demonstrated by in situ hybridization.

OBJECTIVE The purpose was to evaluate the changes in gap junction connexin-43 messenger ribonucleic acid levels associated with rat ovarian follicular development. Gap junctions connect the plasma membranes of adjacent cells through cell-to-cell channels, allowing synchronization of cellular events, including ovarian follicular development. Ovarian gap junctions consist of the protein connexin-43. STUDY DESIGN We used the hypophysectomized immature rat treated with estrogen or gonadotropins as a model to study the ovarian regulation of connexin-43 messenger ribonucleic acid. In situ hybridization with radiolabeled riboprobes was used to localize and quantitate connexin-43 messenger ribonucleic acid. RESULTS We demonstrated that connexin-43 messenger ribonucleic acid was localized to follicular granulosa cells. Estrogen significantly up-regulated connexin-43 messenger ribonucleic acid (91%), whereas gonadotropins that stimulate ovulation and corpus luteum formation completely down-regulated the connexin-43 gene. These results correlate closely with previous immunohistochemical studies of connexin-43 protein. CONCLUSION The positive correlation between follicular development and granulosa cell content of connexin-43 messenger ribonucleic acid is caused by transcriptional activation of the gap junction connexin-43 gene, posttranscriptional stability of connexin-43 messenger ribonucleic acid, or both. Future studies will determine the molecular mechanisms of hormonal regulation of the connexin-43 gene.

Nachweis von GAP Junction Connexin 43-mRNA Veränderung während der ovariellen Follikelreifung durch in situ Hybridisierung

Gap Junctions sind morphologische Membranstrukturen und verbinden als interzellulare Kanale die Plasmamembranen benachbarter Zellen. Sie ermoglichen auf diese Weise die Synchronisation zellularer Aktivitaten (z.B. ovarielle Follikelentwicklung) und den metabolischen Austausch. Die Porengrose betragt ca. 1,5–2 nm und 500–1200 Da Molekule konnen passieren. Gap Junctions werden aus 6 Molekulen des Proteins Connexin (Cx) gebildet, die nach ihrem Molekulargewicht unterschieden werden (4). Eine der Cx Unterarten, Cx 43, kommt bei der Ratte vorwiegend im Myokard vor, findet sich jedoch auch in den Gap Junctions des Ovars und hier vorwiegens in den Granulosazellen (GC9. Das Vorhandensein von Gap Junctions im Follikel last vermuten, das diese morphologischen Membranstrukturen einen GC-zu-GC-Transport von Metabolitetn und Signalmolekulen erlauben und somit eine wichtige Rolle bei der Follikelreifung und der Kontrolle des Wachstums sowie der Reifung der immaturen Oocyte einnehmen (1,2).

Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps**Supported by grant HL 15062 from the National Institutes of Health, Bethesda, Maryland.

OBJECTIVE To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production. DESIGN Contamination of GC by white blood cells was assessed by Wrights stain and immunohistochemistry. Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes [PML]); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes). RESULTS Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4%. Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes. CONCLUSION These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented.