The goal of our laboratory is to elucidate the mechanisms regulating proliferation of smooth muscle cells (SMC), using a concerted biochemical, molecular, and cell biological approach. Hyperproliferation of vascular SMC [Fig. 1] can lead to a wide variety of pathologies, including hypertension and atherosclerosis. SMC hyperproliferation is responsible for the 20-30% failure rates following vascular procedures such as angioplasty and coronary artery bypass grafts.

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Fig. 2 Restenosis following angioplasty. Following a balloon angioplasty, the medial SMC in this rat carotid artery have migrated into the intima and proliferated rapidly to block much of the vessel lumen, a process called restenosis. This cross-section was taken 14 days after the angioplasty, when lesion formation is complete. Alcian Blue was used to stain the vessel wall. IEL = internal elastic lamina.

Aberrant SMC proliferation is also important in non-vascular tissues: uterine fibroids (leiomyomas) are a benign tumor of SMC that affects >20% of all women and >80% of African-American women. More than 200,000 hysterectomies are performed each year in the U.S. due to fibroids, as there is no other surgical or pharmacologic intervention that reduces both symptoms and the recurrence rate.

Several molecules have been described that inhibit the growth of SMC, including heparin [Fig 4]. Earlier work in our laboratory established that the binding of heparin to high affinity binding sites on the cell surface induces alterations in tyrosine phosphorylation and inhibited the activation of Mitogen Activated Protein Kinase (MAPK) and Calcium/ calmodulin-activated protein kinase II (CaMK II), strongly suggesting that heparin mediates its effect through signal transduction pathway(s) that regulate cell proliferation genes.

We undertook the task of identifying heparin-regulated genes that control cell proliferation using both subtractive hybridization and differential display PCR approaches. These studies revealed a novel and potentially important growth arrest-specific gene: CCN5.
Two very important and independent pieces of evidence strongly suggest that CCN5 is an important physiologic regulator of SMC function.
[See Fig. 6 below] CCN5 is abundantly expressed in the uninjured artery wall, but in the injured artery wall (for example, following an angioplasty), CCN5 levels drop dramatically while SMC hyperproliferation occurs and then reappears when the proliferation is complete.

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	Fig. 6 CCN5 expression in the healthy and injured artery wall. Using a highly specific antibody that recognizes CCN5, we see high levels of CCN5 in the medial layers of the healthy rat aorta, in which the SMC are non-proliferating. As shown in the control panels, the IEL and other elastic laminae autofluoresce bright red, making it easy to see the CCN5 staining in between the laminae. Two days after the angioplasty, the SMC are actively traversing the cell cycle, though they have yet to undergo significant cell division. Note the nearly complete absence of CCN5 in these actively proliferating SMC. By 14 days after the injury, the lesion (outlined in white) is complete and the SMC are again non-proliferating. CCN5 is now strongly evident in both the vessel wall and in the lesion.

Further evidence for a pathophysiologic role for CCN5 comes from studies of human fibroids.

click on image for more detail	CCN5 is virtually undetectable in human fibroid tissue, even though it is abundantly expressed in the neighboring normal myometrium. CCN5 inhibits the proliferation and motility of vascular and human uterine SMC, thus suggesting that these cells are potential targets for CCN5-based therapy. [Figs. 8-9].
Fig. 7 CCN5 expression in uterine fibroids. Matched pairs of human fibroid (leiomyoma) tissue and the adjacent normal myometrium from the same uterus were examined for CCN5 levels using Western blot analysis. The phase of the menstrual cycle—proliferating, secretory, or menstrual—was also determined. The actively proliferating fibroid SMC samples express very low levels of CCN5, whereas the normal myometrium samples containing non-proliferating SMC express much higher levels.

We are actively examining the mechanism of action of CCN5 on several levels. Immunolocalization experiments suggest that CCN5 is a “matricellular” protein, i.e., it is secreted by SMC and sticks tightly to the cell surface.
We are also examining the biochemical and molecular mechanism of action using RNA interference to block endogenous expression and viral constructs that permit overexpression of CCN5. Interstingly, CCN5 exhibits matrix-altering ability in that it selectively regulates matrix metalloproteases, an interaction we are actively exploring [Fig. 11].

 

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CCN5 knock-down also results in elevated basal motility, and alterations in the actin cytoskeleton in SMC [Fig. 12-13]. In the uterus, we noticed that CCN5 expression correlated strongly with menstrual cycle phase, with the highest levels found in the proliferative phase, when estrogen levels are highest [Fig. 14].

Fig. 10 CCN5 is secreted and sticks tightly to the cell surface. Using an antibody that specifically recognizes CCN5, we looked at localization of CCN5 in both permeabilized (membrane extracted) and non-permeabilized SMC. CCN5 is red, the actin cytoskeleton is green, and the nucleus is blue. The pattern seen in membrane-extracted cells is highly indicative of a secreted protein, with its strong perinuclear (Golgi) staining and individual small vesicles seen in the cytoplasm. In non-permeabilized cells, CCN5 is not distributed throughout the matrix and instead appears to adhere tightly to the cell surface. This pattern is termed “matricellular”.

To test the hypothesis that estrogen regulates CC5 levels, we used both normal cycling rats and ovariectomized rats [Fig. 15]. In both models, estrogen strongly induces CCN5 expression throughout the uterus [Figs. 16-17].

The data in both vascular and uterine SMC is very consistent [Fig. 18]. Our general model for the role of CCN5 in SMC pathobiology is shown below:

Fig. 19	click on image for more detail

We are pursuing >several important lines of investigation to understand the mechanism of action and functions of CCN5. We are using yeast two-hybrid, gene microarray, image analysis and other cutting-edge methods in these studies [Fig. 20]. A major effort in the laboratory is directed at understanding the pathophysiologic role of CCN5. To this end, we are making knock-out and transgenic mice, and are examining the role of CCN5 in vascular injury models in rats and mice. To attack the role of CCN5 in human fibroids, we are implanting normal and fibroid human SMC that have been engineered to over-express or under-express CCN5 into nude mice, which will permit us to ascertain the ability of CCN5 to reduce human fibroid formation in a small animal model.