Effect of Mechanical Stimulation on Endothelial Phenotype and Gene Expression Profile

Shear Stress-Regulated Gene Expression

Research on the regulation of endothelial gene expression by flow began more than 10 years ago, when expression of specific proteins and transcription of individual genes was compared between static cultures and cells exposed to laminar or disturbed flow. These studies revealed major groups of shear stress-responsive genes, which include transcription factors, cytokines, growth factors, adhesion molecules, enzymes, and cytoskeletal proteins. Analysis of shear responsive promoters identified to date showed that ability to respond to applied shear stresses can be directly linked to the presence of specific, cis-acting sequence elements, termed shear stress responsive elements (SSRE), which were discovered by M. Gimrone's group. Two negative and two positive SSREs within promoter regions of shear stress-regulated genes have been identified so far. In addition, two distinct groups of transcription factors mediate flow-induced induction and suppression of specific gene expression. More recently, endothelial gene regulation by shear stress was documented by several groups using DNA microarray technology [8-11]. These studies compared gene expression profiles in endothelial cells exposed to high-shear steady laminar flow, which represents "athero-protective" flow conditions, and low-shear, nonsteady, nonunidirectional flow (disturbed flow) that simulates conditions in the atherosclerosis-prone areas of the arterial circulation. As result, thousands of endothelial genes were screened, and more than 100 shear stress-regulated genes including genes with not-yet-known function have been detected. The principal conclusions of these studies were as follows: (1) Acute shear stress transiently activates the expression of genes related to the activation of endothelium; (2) chronic laminar shear stress promotes expression of a subset of endothelial genes that can exert potent antithrom-botic, antiadhesive, antiproliferative, and antioxidant effects in endothelial cells; (3) disturbed flow is not simply the absence of laminar flow, but in fact represents a distinct bio-mechanical stimulus that has profound impact upon the gene expression profile of endothelial cell culture; and (4) under chronic (24 hours) conditions of laminar flow, more genes are suppressed than induced, whereas disturbed flow dramatically induces proatherogenic, proinflammatory genes such as E-selectin, VCAM-1, ICAM1, PECAM-1, throm-bospondin, and MCP-1. These findings support the notion about a specific shear stress-mediated EC phenotype that may play an important role in the development of vascular pathologies and responsiveness of the EC monolayer to external chemical and mechanical stimuli.

Cyclic Stretch-Regulated Gene Expression

Similar to flow, mechanical strain is also a potent regulator of gene expression. Phenotypic responses of vascular cells exposed to cyclic stretch in vitro include increased

Table I Selected Genes Differentially Regulated by Low- and High-Amplitude Cyclic Stretch.

Fold change Fold change at 5% at 18%

Signal transduction

Inducible T-cell kinase

17.1

Nuclear receptor subfamily 1, group D, member 2

3.2

Angiopoietin 2

1.8

2.1

HMG-CoA-synthase

NC

4.0

Proteinase-activated receptor 2

1.4

2.1

Proteinase-activated receptor 1

NC

-1.2

Rho B GTPase

1.87

2.1

Rho C GTPase

NC

1.4

Cell adhesion

Gap junction protein, alpha 5

2.8

Intercellular adhesion molecule 1

NC

2.0

(CD54)

Beta 3 Integrin

1.5

2.1

Beta-catenin

NC

2.1

Cadherin-13

8.5

1.7

Cell—cell signaling

Placental growth factor,

1.8

2.0

VEGF-related

Ephrin A1

1.2

2.1

Ephrin B2

1.7

Smooth muscle myosin heavy chain

NC

1.4

Filamin

NC

1.7

Inflammation/remodeling

Human cyclooxygenase-2

NC

3.0

TGF-b superfamily protein

1.6

2.3

Proteinase-activated receptor 2

1.4

2.1

ZIP-kinase

1.3

1.7

Expression profiling was performed using the Affymetrix GeneChip system. Samples obtained from cells exposed to 5 percent or 18 percent cyclic stretch (48 hours) were hybridized to the Affymetrix HGU95Av2 Array (~12,000 full-length genes). Affymetrix Microarray Suite software was used to determine relative gene expression. GeneSpring and MAPPFinder software was used for microarray data analysis. Results represent fold increase in cDNA signal in cyclic stretch-preconditioned cells over static control. NC, no change; —, signal absent.

Expression profiling was performed using the Affymetrix GeneChip system. Samples obtained from cells exposed to 5 percent or 18 percent cyclic stretch (48 hours) were hybridized to the Affymetrix HGU95Av2 Array (~12,000 full-length genes). Affymetrix Microarray Suite software was used to determine relative gene expression. GeneSpring and MAPPFinder software was used for microarray data analysis. Results represent fold increase in cDNA signal in cyclic stretch-preconditioned cells over static control. NC, no change; —, signal absent.

SHEAR STRESS

CYCLIC STRETCH

DISTURBED, ACUTE PATHOLOGICAL

CHRONIC LAMINAR CHANGES IN LAMINAR FLOW PHYSIOLOGICAL (INCREASED)

- ATHEROPROTECTION - INFLAMMATION

- STABILIZATION OF ENDOTHELIAL BARRIER - ATHEROGENESIS

- VASCULAR REMODELING

- ENDOTHELIAL BARRIER COMPROMISE

Figure 2 The differential responses of the endothelium to various patterns of shear stress and cyclic stretch. Physiologically relevant laminar shear stress and cyclic stretch promote endothelial cell quiescent state, induce expression of antiatherogenic and anti-inflammatory genes, and promote endothelial barrier integrity and recovery after edemagenic stimuli such as thrombin. In contrast, disturbed flow and pathologically increased amplitudes of cyclic stretch induce endothelial activation, extracellular matrix production, and expression of proinflammatory and apoptotic genes, which may lead to vascular remodeling and propagate endothelial barrier dysfunction, atherogenesis, and inflammatory processes in the vasculature.

expression of contractile and cytoskeletal proteins (myosin light chain kinase, smooth muscle myosin heavy chains, desmin, h-caldesmon) and increased expression of thrombin receptor PARI in vascular smooth muscle cells. A number of bioactive proteins regulated by cyclic stretch have been also identified in endothelial cells and macrophages and include IL-8, TGF-beta, VEGF, and monocyte chemotactic protein-1. Analysis of vascular gene expression regulated by mechanical strain reveals differential responses to physiological and pathophysiological (increased) levels of mechanical strain. For example, release of FGF-2, a growth factor involved in cellular repair after injury, is induced in vascular smooth muscle cells stretched at 14 percent and 33 percent elongation, but not at 5 percent elongation. Significant increase in IL-8 production is observed in endothelial cells exposed to cyclic stretch at 15 percent elongation, whereas stretch at 6 percent elongation did not affect IL-8 levels. In pulmonary circulation, pathological overdistension of the lung may induce inflammatory process-triggered mechanical activation of macrophages and epithelial and endothelial cells, which may cause alveolar and endothelial barrier dysfunction and vascular leak and culminate in pulmonary edema. Recently, microarray DNA technologies have been applied to assess time and amplitude dependence of cyclic stretch effects on gene expression profile in human pulmonary endothelial cells [2]. The results showed that cyclic stretch at physiologically relevant and pathological amplitudes (5% and 18% elongation, respectively) induced distinct expression patterns of genes involved in signal transduction, cytoskeletal remodeling, cell adhesion, inflammatory responses, and regulation of endothelial barrier function (Table I).

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