[Frontiers in Bioscience 3, d194-207, February 15, 1998]

	[Frontiers in Bioscience 3, d194-207, February 15, 1998] Reprints PubMed CAVEAT LECTOR
	VASOPRESSIN SIGNALING PATHWAYS IN VASCULAR SMOOTH MUSCLE Raphael A. Nemenoff Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262 Received 1/21/98 Accepted 2/4/98 3. PHYSIOLOGIC EFFECTS OF VASOPRESSIN IN SMOOTH MUSCLE Cultures of vascular smooth muscle cells have been extensively employed as a model for studying the physiologic and pathophysiologic responses of vascular smooth muscle in vivo (13, 14). These preparations have been characterized by monitoring expression of smooth muscle specific genes such as the smooth muscle isoform of alpha-actin (SM- alpha-actin) or smooth muscle myosin (15). In general, freshly isolated primary cultures from adult vessels are slow proliferating and manifest features of highly differentiated vascular smooth muscle. These cells can be subcultured and retain many vascular smooth muscle features for multiple passages. However, eventually they will begin to grow more rapidly and lose many of the characteristics of the differentiated phenotype. In fact, by growing cells, we are selecting for proliferative phenotypes. Most workers have therefore performed experiments in early passage, using some arbitrary cutoff. Studies performed in our laboratory have generally used cells subcultured for less than 8 passages. Most workers using such preparations have isolated cells from large conductance vessels such as aorta. It is likely that VSMC isolated from resistance vessels will exhibit distinct physiologic responses. Acute stimulation of early passage VSMC by AVP as well as angiotensin II results in contraction (16, 17, 18). Many groups have studied contraction in cultured cells by measuring shape change following agonist stimulation. While this is easily quantitated, the relationship of a change in shape of a cell attached to a plastic substratum to contraction of a vessel is not clear. Growth of VSMC on polymerized silicone-coated dishes allows measurement of contractile force by the appearance of wrinkles (19). However, this technique is more difficult to quantitate. Care also needs to be taken that growth of these cells on silicone dishes does not induce phenotypic changes in the cells, making it difficult to compare signaling studies done under standard tissue culture conditions. Long-term exposure of VSMC to AVP in the absence of other mitogens, or in the presence of low concentrations of serum, results in an increase in protein content per cell, which we will define as hypertrophy (20, 21). This induction requires several days of continuous exposure to AVP, and is also observed with other vasoconstrictors such as angiotensin II (22). No increases in DNA synthesis as assessed by ³H-thymidine incorporation into DNA is observed, and there is no increase in cell number. At higher concentrations of serum, or in the presence of other mitogens, AVP potentiates DNA synthesis and cell proliferation, acting as a progression factor. These results can be contrasted with exposure of VSMC to growth factors such as PDGF, which as a sole factor causes increases 3H-thymidine uptake into DNA and promotes mitogenesis, characteristic of a competence factor. We have recently examined the effects of AVP on progression of VSMC through the cell cycle using flow cytometry. AVP stimulation resulted in cells which accumulated in G1, and did not enter S phase². Perhaps suprisingly, AVP and PDGF stimulate many of the same early post-receptor signaling events. These include increases in intracellular Ca²⁺, activation of protein kinases and phospholipases, and induction of immediate early response genes. The factors mediating the distinct hypertrophic response seen with AVP versus the proliferative response of PDGF is therefore not clear. We propose three conceptual models to account for these findings (figure 1). One possibility is that AVP only gives a partial mitogenic signal. In this model, PDGF would engage some specific effector(s), critical for mitogenesis, which is not activated by AVP (figure 1A). Alternatively, it has been proposed that vasoconstrictors induce both a mitogenic and anti-mitogenic signal (figure 1B). To that end it has been shown that angiotensin II induces the expression of TGF-beta in VSMC, which has antimitogenic effects (23). The combination of the two signals (mitogenic and anti-mitogenic) would be predicted to result in hypertrophy of the cells. Finally, a third model would propose that vasoconstrictors such as AVP engage distinct signaling pathways not controlled by PDGF which lead to hypertrophy (figure 1C). We and others have recently Figure 1. Models of AVP Action in VSMC. We propose three specific models to account for the hypertrophic response seen with AVP, as opposed to the proliferative response seen with PDGF. In the first model (Panel A), PDGF stimulates three hypothetical pathways (X,Y, and Z) required for proliferation. AVP, through a G protein only stimulates 2 of these (X and Y), leading to an incomplete mitogenic signal. In model 2 (Panel B), AVP stimulates the same three hypothetical mitogenic effectors as PDGF, but in addition also stimulates production of an anti-mitogenic agent, in this case TGF-b. The simultaneous engagement of mitogenic and anti-mitogenic pathways results in hypertrophy. In model 3 (Panel C), AVP stimulates a mitogenic response plus induces differentiation of the cells into a more highly differentiated contractile phenotype, as indicated by the induction of SM-alpha-actin (SM-alpha-act.). This combination of proliferation and conversion to a non-proliferative phenotype results in hypertrophy. reported that AVP as well as angiotensin II increase the expression of muscle-specific genes such as the smooth muscle isoform of alpha-actin (SM-alpha-actin). This effect is mediated through increased transcription of this gene (see below). SM-alpha-actin has been used as a marker for the state of differentiation of VSMC (15). Developmentally, expression is low in embryonic and developing vessels, and increases as the vessels mature and the cells convert from a proliferative to a contractile phenotype. In the setting of atherosclerosis, VSMC undergo a "de-differentiation" to a more proliferative phenotype and this is associated with suppression of SM-alpha-actin expression. Importantly, exposure of VSMC to PDGF suppresses expression of SM-alpha-actin, and is able to inhibit induction in response to vasoconstrictors (24). Thus the regulation of SM-alpha-actin expression represents a physiologic action which is regulated in opposite directions by vasoconstrictors and PDGF. A simplistic model would propose that AVP or angiotensin II result in a mitogenic stimulation together with a signal promoting differentiation of the cells to a contractile, non-proliferative phenotype (High SM-alpha-actin), whereas PDGF results in a mitogenic signal combined with promotion to a proliferative phenotype (low SM-alpha-actin). Delineating the upstream signaling events regulating SM-alpha-actin expression may therefore provide a better understanding of the specific signaling pathways engaged by AVP, leading to hypertrophy. Finally, it should be emphasized that these three models proposed are not mutually exclusive. To that end it has been reported that TGF-beta induces hypertrophy in VSMC (25). More recently a number of established cell lines derived from vascular smooth muscle cells by immortilization techniques have become available. These include the A7r5 and A10 cell lines. While these cells are responsive to AVP (26), and have been useful in defining early post-receptor events, care must be taken in extrapolating results obtained with these cell lines to the biology of vascular smooth muscle in vivo. In particular, we have noted that chronic effects of AVP observed in early passage rat aortic VSMC are not reproduced in either of these cell lines². AVP-induced hypertrophy and induction of smooth muscle alpha-actin expression did not occur in either cell line. Furthermore, phenotypic modulation achieved by growing VSMC on specific extracellular matrices is also not achieved in these cells. Growth of early passage VSMC on Matrigel, an extracellular matrix preparation rich in laminin promotes conversion to the contractile phenotype (27), which is characterized by elevated SM-alpha-actin expression. Established cell lines fail to mimic this response. Thus data on specific signaling pathways obtained in these cell lines needs to be confirmed in a more "physiologic" preparation. With the cloning of many signaling enzymes and effectors, a powerful technique to study post-receptor signaling has been the use of cell transfection experiments. Expressing mutations encoding either constitutively active or dominant-negative mutants of individual signaling molecules has been widely employed in other cell types such as fibroblasts. These studies enable investigators to examine the role of signaling pathways for which specific pharmacological agents are not currently available. The development of stable cell lines expressing foreign genes requires growth of individual clones in selectable media. This process entails many cell divisions, and raises the concern that transfection of VSMC will lead to dedifferentiation and loss of vascular smooth muscle features. In our studies we have successfully used cells transfected with a plasmid lacking a cDNA insert as a control for the transfection process (28, 29). As with all techniques, use of molecular biological approaches is not a panacea. Effects of expression of foreign genes in these cells can be antagonized by the cells, through activation of compensatory mechanisms. This problem can be addressed using both stable and transient expression of foreign genes, or by placing them under the control of inducible promoters.

[Frontiers in Bioscience 3, d194-207, February 15, 1998]
Reprints
PubMed
CAVEAT LECTOR

VASOPRESSIN SIGNALING PATHWAYS IN VASCULAR SMOOTH MUSCLE

Raphael A. Nemenoff

Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262

Received 1/21/98 Accepted 2/4/98

3. PHYSIOLOGIC EFFECTS OF VASOPRESSIN IN SMOOTH MUSCLE

Cultures of vascular smooth muscle cells have been extensively employed as a model for studying the physiologic and pathophysiologic responses of vascular smooth muscle in vivo (13, 14). These preparations have been characterized by monitoring expression of smooth muscle specific genes such as the smooth muscle isoform of alpha-actin (SM- alpha-actin) or smooth muscle myosin (15). In general, freshly isolated primary cultures from adult vessels are slow proliferating and manifest features of highly differentiated vascular smooth muscle. These cells can be subcultured and retain many vascular smooth muscle features for multiple passages. However, eventually they will begin to grow more rapidly and lose many of the characteristics of the differentiated phenotype. In fact, by growing cells, we are selecting for proliferative phenotypes. Most workers have therefore performed experiments in early passage, using some arbitrary cutoff. Studies performed in our laboratory have generally used cells subcultured for less than 8 passages. Most workers using such preparations have isolated cells from large conductance vessels such as aorta. It is likely that VSMC isolated from resistance vessels will exhibit distinct physiologic responses.

Acute stimulation of early passage VSMC by AVP as well as angiotensin II results in contraction (16, 17, 18). Many groups have studied contraction in cultured cells by measuring shape change following agonist stimulation. While this is easily quantitated, the relationship of a change in shape of a cell attached to a plastic substratum to contraction of a vessel is not clear. Growth of VSMC on polymerized silicone-coated dishes allows measurement of contractile force by the appearance of wrinkles (19). However, this technique is more difficult to quantitate. Care also needs to be taken that growth of these cells on silicone dishes does not induce phenotypic changes in the cells, making it difficult to compare signaling studies done under standard tissue culture conditions.

Long-term exposure of VSMC to AVP in the absence of other mitogens, or in the presence of low concentrations of serum, results in an increase in protein content per cell, which we will define as hypertrophy (20, 21). This induction requires several days of continuous exposure to AVP, and is also observed with other vasoconstrictors such as angiotensin II (22). No increases in DNA synthesis as assessed by ³H-thymidine incorporation into DNA is observed, and there is no increase in cell number. At higher concentrations of serum, or in the presence of other mitogens, AVP potentiates DNA synthesis and cell proliferation, acting as a progression factor. These results can be contrasted with exposure of VSMC to growth factors such as PDGF, which as a sole factor causes increases 3H-thymidine uptake into DNA and promotes mitogenesis, characteristic of a competence factor. We have recently examined the effects of AVP on progression of VSMC through the cell cycle using flow cytometry. AVP stimulation resulted in cells which accumulated in G1, and did not enter S phase².

Perhaps suprisingly, AVP and PDGF stimulate many of the same early post-receptor signaling events. These include increases in intracellular Ca²⁺, activation of protein kinases and phospholipases, and induction of immediate early response genes. The factors mediating the distinct hypertrophic response seen with AVP versus the proliferative response of PDGF is therefore not clear. We propose three conceptual models to account for these findings (figure 1). One possibility is that AVP only gives a partial mitogenic signal. In this model, PDGF would engage some specific effector(s), critical for mitogenesis, which is not activated by AVP (figure 1A). Alternatively, it has been proposed that vasoconstrictors induce both a mitogenic and anti-mitogenic signal (figure 1B). To that end it has been shown that angiotensin II induces the expression of TGF-beta in VSMC, which has antimitogenic effects (23). The combination of the two signals (mitogenic and anti-mitogenic) would be predicted to result in hypertrophy of the cells. Finally, a third model would propose that vasoconstrictors such as AVP engage distinct signaling pathways not controlled by PDGF which lead to hypertrophy (figure 1C). We and others have recently

Figure 1. Models of AVP Action in VSMC. We propose three specific models to account for the hypertrophic response seen with AVP, as opposed to the proliferative response seen with PDGF. In the first model (Panel A), PDGF stimulates three hypothetical pathways (X,Y, and Z) required for proliferation. AVP, through a G protein only stimulates 2 of these (X and Y), leading to an incomplete mitogenic signal. In model 2 (Panel B), AVP stimulates the same three hypothetical mitogenic effectors as PDGF, but in addition also stimulates production of an anti-mitogenic agent, in this case TGF-b. The simultaneous engagement of mitogenic and anti-mitogenic pathways results in hypertrophy. In model 3 (Panel C), AVP stimulates a mitogenic response plus induces differentiation of the cells into a more highly differentiated contractile phenotype, as indicated by the induction of SM-alpha-actin (SM-alpha-act.). This combination of proliferation and conversion to a non-proliferative phenotype results in hypertrophy.

reported that AVP as well as angiotensin II increase the expression of muscle-specific genes such as the smooth muscle isoform of alpha-actin (SM-alpha-actin). This effect is mediated through increased transcription of this gene (see below). SM-alpha-actin has been used as a marker for the state of differentiation of VSMC (15). Developmentally, expression is low in embryonic and developing vessels, and increases as the vessels mature and the cells convert from a proliferative to a contractile phenotype. In the setting of atherosclerosis, VSMC undergo a "de-differentiation" to a more proliferative phenotype and this is associated with suppression of SM-alpha-actin expression. Importantly, exposure of VSMC to PDGF suppresses expression of SM-alpha-actin, and is able to inhibit induction in response to vasoconstrictors (24). Thus the regulation of SM-alpha-actin expression represents a physiologic action which is regulated in opposite directions by vasoconstrictors and PDGF. A simplistic model would propose that AVP or angiotensin II result in a mitogenic stimulation together with a signal promoting differentiation of the cells to a contractile, non-proliferative phenotype (High SM-alpha-actin), whereas PDGF results in a mitogenic signal combined with promotion to a proliferative phenotype (low SM-alpha-actin). Delineating the upstream signaling events regulating SM-alpha-actin expression may therefore provide a better understanding of the specific signaling pathways engaged by AVP, leading to hypertrophy. Finally, it should be emphasized that these three models proposed are not mutually exclusive. To that end it has been reported that TGF-beta induces hypertrophy in VSMC (25).

More recently a number of established cell lines derived from vascular smooth muscle cells by immortilization techniques have become available. These include the A7r5 and A10 cell lines. While these cells are responsive to AVP (26), and have been useful in defining early post-receptor events, care must be taken in extrapolating results obtained with these cell lines to the biology of vascular smooth muscle in vivo. In particular, we have noted that chronic effects of AVP observed in early passage rat aortic VSMC are not reproduced in either of these cell lines². AVP-induced hypertrophy and induction of smooth muscle alpha-actin expression did not occur in either cell line. Furthermore, phenotypic modulation achieved by growing VSMC on specific extracellular matrices is also not achieved in these cells. Growth of early passage VSMC on Matrigel, an extracellular matrix preparation rich in laminin promotes conversion to the contractile phenotype (27), which is characterized by elevated SM-alpha-actin expression. Established cell lines fail to mimic this response. Thus data on specific signaling pathways obtained in these cell lines needs to be confirmed in a more "physiologic" preparation.

With the cloning of many signaling enzymes and effectors, a powerful technique to study post-receptor signaling has been the use of cell transfection experiments. Expressing mutations encoding either constitutively active or dominant-negative mutants of individual signaling molecules has been widely employed in other cell types such as fibroblasts. These studies enable investigators to examine the role of signaling pathways for which specific pharmacological agents are not currently available. The development of stable cell lines expressing foreign genes requires growth of individual clones in selectable media. This process entails many cell divisions, and raises the concern that transfection of VSMC will lead to dedifferentiation and loss of vascular smooth muscle features. In our studies we have successfully used cells transfected with a plasmid lacking a cDNA insert as a control for the transfection process (28, 29). As with all techniques, use of molecular biological approaches is not a panacea. Effects of expression of foreign genes in these cells can be antagonized by the cells, through activation of compensatory mechanisms. This problem can be addressed using both stable and transient expression of foreign genes, or by placing them under the control of inducible promoters.