Eugene A. Sprague

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

Vascular endottielial cell proliferation in culture and the influence of flow

The influence of laminar shear stress on cell proliferation was investigated for subconfluent bovine aortic endothelial cell monolayers seeded on either glass or Thermanox. The effect of both steady and pulsatile shear stress was studied. For bovine aortic endothelial cells on Thermanox exposed to steady flow, shear stress levels greater than 15 dyne/cm2 resulted in a dose-related reduction in the rate of cell proliferation. At 90 dyne/cm2, the rate of proliferation was virtually totally arrested for 48 h, but then resumed. Pulsatile shear stress produced an exaggeration of the effect observed in response to steady shear stress. Bovine aortic endothelial cells seeded on glass, exhibited a similar but more sensitive response, with a significant reduction in growth rate observed after 24 h at shear stress levels greater than 5 dyne/cm2 and a near cessation of proliferation at 13 dyne/cm2.

Influence of a laminar steady-state fluid-imposed wall shear stress on the binding, internalization, and degradation of low-density lipoproteins by cultured arterial endothelium.

Fluid mechanical steady-state laminar wall shear stresses of 30 dyne/cm2 (high stress) and less than 1 dyne/cm2 (low stress) have been applied for varying times to confluent cultures of bovine aortic endothelial cells (BAECs) by means of two parallel plate channel flow chambers in series. BAEC cultures not exposed to shear or flow (no stress) were also studied. A shear stress of 30 dyne/cm2 resulted in cellular elongation and alignment, changes that were largely complete by 24 hr. In experiments in which BAECs were incubated with 125I-labeled low-density lipoprotein for 2 or 24 hr in the presence of shear stress levels, 125I-LDL internalization at 24 hr was increased (p less than .05) in response to high-stress conditions. This increased uptake of 125I-LDL was observed in BAECs prealigned for 24 hr under high stress and in BAECs undergoing alignment in the presence of circulating 125I-LDL. BAECs were also exposed to shear stress for 24 hr in the presence of a lipoprotein-deficient circulating medium to maximize LDL receptor expression. Receptor-mediated 125I-LDL internalization and degradation measured immediately after shear stress were both significantly enhanced (p less than .01) in BAECs exposed to high stress. Furthermore, 125I-LDL binding studies at 4 degrees C revealed a significant increase (p less than .01) in specific 125I-LDL binding to BAECs exposed to high stress relative to those exposed to low or no stress. Nonspecific 125I-LDL endocytosis was not influenced by shear stress levels.(ABSTRACT TRUNCATED AT 250 WORDS)

A modern view of atherogenesis

Two key events in the atherogenic cascade are the focal influx and accumulation of low-density lipoprotein (LDL) cholesterol at arterial sites having a predilection for atherosclerotic lesion development and the recruitment of blood monocytes to these lesion-prone sites. Both processes are enhanced in the setting of hyperlipidemia and dyslipoproteinemia. The monocytes recruited to the endothelial surface subsequently migrate to the subendothelial space under the directed guidance of chemoattractants, such as monocyte chemotactic protein-1 and oxidatively modified LDL. These cells then undergo activation-differentiation to become macrophages. At the same time, LDL, and probably other lipoproteins such as the small dense LDL particles and lipoprotein (a), traverse the endothelium and undergo oxidative modification by reactive oxygen species. These oxidatively modified lipoproteins are recognizable by the non-down-regulating macrophage scavenger receptor. Their uptake by these receptors results in the formation of the foam cell characteristic of early-stage atherosclerosis. As monocyte recruitment and lipoprotein influx continue, the lesion grows and develops into the fatty streak. Subsequent foam cell necrosis due to the influence of cytotoxic oxidatively modified LDL and increased collagen synthesis by intimal smooth muscle cells lead to the established atherosclerotic lesion referred to as the fibrous plaque. As our understanding of the mechanisms involved in the pathogenesis of atherosclerosis has evolved over the past few years, novel strategies for intervention in the atherogenic process have emerged.

Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte-Like MLO-Y4 Cells†

Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte‐like cell line MLO‐Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO‐Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO‐Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO‐Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. “Scrape‐loading” dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte‐like cells was not observed in osteoblast‐like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte‐like MLO‐Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.

PGE2 Is Essential for Gap Junction-Mediated Intercellular Communication between Osteocyte-Like MLO-Y4 Cells in Response to Mechanical Strain

We have observed, in our previous studies, that fluid flow increases gap junction-mediated intercellular coupling and the expression of a gap junction protein, connexin 43, in osteocyte-like MLO-Y4 cells. Interestingly, this stimulation is further enhanced during the poststress period, indicating that a released factor(s) is likely to be involved. Here, we report that the conditioned medium obtained from the fluid flow-treated MLO-Y4 cells increased the number of functional gap junctions and connexin 43 protein. These changes are similar to those observed in MLO-Y4 cells directly exposed to fluid flow. Fluid flow was found to induce PGE(2) release and increase cyclooxygenase 2 expression. Treatment of the cells with PGE(2) had the same effect as fluid flow, suggesting that PGE(2) could be responsible for these autocrine effects. When PGE(2) was depleted from the fluid flow-conditioned medium, the stimulatory effect on gap junctions was partially, but significantly, decreased. Addition of the cyclooxygenase inhibitor, indomethacin, partially blocked the stimulatory effects of mechanical strain on gap junctions. Taken together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated, in part, by the release of PGE(2). Hence, PGE(2) is an essential mediator between mechanical strain and gap junctions in osteocyte-like cells.

Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

Pathogenesis of the Atherosclerotic Lesion: Implications for diabetes mellitus

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents—the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

Influence of Surface Topography on Endothelialization of Intravascular Metallic Material

PURPOSE To determine whether grooves on a metal surface help endothelialization and, furthermore, what groove size is more likely to promote the fastest endothelialization in an in vitro model. Hypothetically, a microscopic pattern of parallel grooves disposed in the direction of flow, on the inner surface of stents, increases endothelial cell migration rates, resulting in decreased time to total coverage of the prosthetic surface. MATERIALS AND METHODS Square, flat pieces of nitinol were placed level on a monolayer, confluent culture of endothelial cells. The metal pieces were treated to produce parallel grooves on the surface of 1, 3, 15, and 22 microm to be compared to polished, smooth controls. Microscopy images were obtained by digital capture and processed for analysis of migration distance and cell count, density, shape, and alignment. RESULTS Grooved surfaces promoted increased rate of migration of endothelial cells, up to 64.6% when compared to smooth, control surfaces. Larger grooves resulted in greater migration rates. The cells aligned with the grooves, elongated, and become more numerous on grooved surfaces, particularly with large grooves. CONCLUSION A pattern of microscopic parallel grooves more than doubles the migration rate of endothelial cells over metallic surfaces ordinarily used for endovascular stents. Future research in this area is aimed at demonstrating the potential effect of grooved endovascular stent surfaces on faster endothelialization times.

Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading

Bone tissues respond to mechanical loading/unloading regimens to accommodate (re)modeling requirements; however, the underlying molecular mechanism responsible for these responses is largely unknown. Previously, we reported that connexin (Cx) 43 hemichannels in mechanosensing osteocytes mediate the release of prostaglandin, PGE2, a crucial factor for bone formation in response to anabolic loading. We show here that the opening of hemichannels and release of PGE2 by shear stress were significantly inhibited by a potent antibody we developed that specifically blocks Cx43-hemichannels, but not gap junctions or other channels. The opening of hemichannels and release of PGE2 are magnitude-dependent on the level of shear stress. Insertion of a rest period between stress enhances this response. Hemichannels gradually close after 24 h of continuous shear stress corresponding with reduced Cx43 expression on the cell surface, thereby reducing any potential negative effects of channels staying open for extended periods. These data suggest that Cx43-hemichannel activity associated with PGE2 release is adaptively regulated by mechanical loading to provide an effective means of regulating levels of extracellular signaling molecules responsible for initiation of bone (re)modeling.