In Vitro Cell.Dev.Biol.Animal DOI 10.

1007/s11626-011-9430-7

REPORT

Characterization of Clonal Vascular Smooth Muscle Cell Lines Derived from Y oung and Old Fischer 344 Rats
William E. Schutzer & Douglas R. Beard & John F. Reed & Scott L. Mader

Received: 22 February 2011 / Accepted: 19 May 2011 / Editor: T. Okamoto # The Society for In Vitro Biology 2011

Abstract A significant finding with aging humans (and aging animal models) is that blood vessels lose their ability to respond to beta-adrenergic receptor stimuli. Therefore, they produce less cyclic adenosine monophosphate (cAMP) and have decreased vasorelaxation with advancing age. This change likely contributes to hypertension, insufficient blood flow, and atherosclerosis. Our goal was to develop a vascular smooth muscle cell culture model that replicates the molecular and biochemical changes observed in blood vessels with advancing age. A clonal selection strategy was used to produce cell lines from 2-, 6-, 12-, and 24-month-old male Fischer 344 rat aortae. Cultures were validated as smooth muscle cells with immunocytochemistry positive for -actin and negative for von Willebrand factor VIII. Positive staining for G protein-coupled receptor kinase 2 indicated presence of this adrenergic receptor regulator. A total of n =5 clones from n =7 animals for each age group were initially analyzed for cAMP accumulation under three conditions: basal, isoproterenol stimulated, and forskolin stimulated. Results found that at passage 3, there was a significant reduction in cAMP accumulation to isoproterenol. However, this reduction disappeared by passage 6. Secondary analysis segregated clones into phenotypic age groups independent of donor animal age. Segregation identified n =3 clones per group. At passage 3, the age-related change in the betaW. E. Schutzer : D. R. Beard : J. F. Reed : S. L. Mader (*) Portland V A Medical Center, Research Service, R&D 26, 3710 SW US V eterans Hospital Rd., Portland, OR 97201, USA e-mail: scott.mader@va.gov W. E. Schutzer : S. L. Mader School of Medicine, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239-3098, USA

adrenergic change was magnified. However, even with segregation, the adrenergic response was lost by passage 6. Our results show that early passaged clonal vascular smooth muscle cell cultures maintain their aging, adrenergic phenotype. Two separate strategies to identify age-representative phenotypes into later passage were unsuccessful. Keywords Aging . Adrenergic, beta . Fischer 344 . Clonal Selection . cAMP. Cell culture . Isoproterenol . V ascular . G protein-coupled receptor kinase One well-documented biochemical change found in the aging vasculature is diminished beta-adrenergic receptor (-AR)-mediated cyclic adenosine monophosphate (cAMP) production, and thus vasorelaxation. This change, commonly observed in elderly patients, is thought to contribute to the clinical observations of hypertension, orthostatic hypotension, and arterial insufficiency (Schutzer and Mader 2003). The molecular, mechanistic cause for this change is not known, and studies to this end are problematic with intact tissue. However, intensive mechanistic and molecular studies to examine this change could be efficiently and accurately conducted in cultured vascular smooth muscle cells (VSMC). To our knowledge, the initial attempt to culture VSMC specifically for the purpose of examining age-related changes in -AR signaling was performed by V olicer et al. (1983), and these investigators found an age-related decrease in epinephrine-mediated cAMP production. However, attempts to replicate these results have not been successful (Mader 1992; Chin and Hoffman 1991). One report (Crass et al. 1992) determined that primary cultures derived from aortic VSMC maintained an aging phenotype in regard to the -AR response. However, as

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those cultures were passaged, the aging/-AR phenotype disappeared. Because our research is focused on understanding the causes of the age-related decline in -AR-mediated signaling, with an ultimate goal of providing a therapeutic target for intervention, we attempted to generate a stable cultured cell line that mirrors what is observed in intact vascular tissue across age groups. One possible reason that previous attempts failed to produce cultured VSMCs that maintained the aging phenotype with continued passage may be because those cultures were generated with standard techniques and thus contained multiple founder cells that would likely yield cultures with cells that expressed heterogeneous phenotypes. One way to eliminate this possible variability is use clonal selection. Rothman et al. (1992) used this technique to produce pulmonary arterial clone 1 (PAC1) cells that provided a standardized and homogenous population of cells that have been used to understand mechanisms associated with pulmonary hypertension (Button et al. 1994; Rothman et al. 1994; Lu et al. 1996, 1998). To date, no defined cultured VSMC line exists that maintains the -AR-specific aging phenotype. Herein, we characterize the adrenergic responses of cultured VSMC lines derived from male Fischer 344 rats aged 2-, 6-, 12-, and 24-m-old generated by clonal selection. The primary outcome variable was the effect of isoproterenol (ISO; a -AR agonist)- and forskolin (FSK; an adenylate cyclase activator)-mediated cAMP accumulation. There is a wealth of literature documenting the agerelated decline in -AR-mediated cAMP production in vascular smooth muscle (V olicer et al. 1983; Chin and Hoffman 1991; Crass et al. 1992; Mader 1992; Chin et al. 1996). Two approaches were used to process the results. The first strategy was to derive clones from each of the respective age groups. The second strategy recognized the documented heterogeneity among vascular smooth muscle cells and thus ignored the donor animals age and segregated clones into age groups that were based on defined age criteria (presented following). The groups were young, mature, old, and old-old. We evaluated our clonal cells at passages 3 and 6. The passage 3 point was chosen because this was the first opportunity that enough clonal cells were available for cAMP analysis: one 100-mm dish (see following for a description of the generation of the clonal cultures). The second evaluation was at passage 6 to verify the ability to maintain phenotype. This was considered important as this was the first opportunity that enough cells (multiple 100 mm dishes) would be available to perform the molecular mechanistic comparisons among multiple treatments (see for example Schutzer et al. 2000). Clonal cell lines were derived from thoracic aortae collected from male Fischer 344 rats aged 2-, 6-, 12-, or 24-months old (Harlan SpragueDawley; NIA colony).

Aortae were split lengthwise, the endothelium was removed with gentle rubbing, divided into approximately 10 mm2 pieces and adhered to 100 mm plates with the medial layer down (one plate each per animal). Upon adherence, Dulbecco's modified Eagle's medium (DMEM) with penicillin and streptomycin (Invitrogen [Gibco], Carlsbad, CA) was added, and these primary cultures were grown for 10 d, with daily inspection for VSMC proliferation and smooth muscle morphology (hillock-and-valley pattern of cultures, bipolar and/or ribbon-shaped individual cells, multi-nucleoli per nucleus). These primary cultures were removed by trypsin digestion and were replated sparsely (125 cells/cm2) in conditioned media (1:1 fresh DMEM with filtered DMEM collected from previous VSMC cultures). These passage 1 cells were allowed to incubate overnight and the attachment locations of individual cells were marked on the plate as identified with phase-contrast light microscopy. These cells were evaluated daily to ensure that subsequent colonies were derived from the initially identified founder cell. Nine days beyond, these colonies were collected by surrounding each colony with a glass cloning ring and trypsin digestion. These passage 2 cells from each clonal colony were re-plated at a density of 500 cells/cm2 in 100 mm dishes, and allowed to proliferate for 2 wk. To examine the properties of each clonal line, cells were trypsinized, and re-plated (passage 3) into: (a) 24-well plates cAMP analysis (response to ISO and FSK), (b) cover slips for immunocytochemistry (smooth muscle specific -actin, endothelial cell specific anti-von Willebrand factor VIII, and G protein receptor kinase 2), (c) 100 mm plates that were used to seed passage 46 experiments, or (d) frozen in liquid N2. Prior to evaluating clonal response to -AR activation, cultures were validated as smooth muscle, versus endothelial cells or fibroblasts, with immunocytochemistry (ICC) for smooth muscle specific -actin, and von Willebrand factor VIII. The -actin protein is among the most frequently used markers for identification of VSMC, and this protein is expressed in high concentrations in the contractile phenotypic VSMC but is expressed in low concentrations in the synthetic phenotypic VSMC (Song et al. 2001). The von Willebrand factor protein is one of the most widely used endothelial cell markers in blood vessels for the identification of endothelial lineage (Xu et al. 2009). Figure 1A shows positive staining for -actin as well as the characteristic striated pattern of localization, thus confirming that our cultures were VSM in the contractile state. Also, all cultures were negative for von Willebrand factor VIII (not shown) suggesting that the cultures were free from contamination of endothelial cells. Finally, ICC for G protein receptor kinase 2 (GRK2) was conducted to verify presence of this major regulator of the adrenergic signaling axis (Pitcher et al. 1998). Figure 1B shows

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Figure 1. Immunocytochemistry of clonal vascular smooth muscle cells demonstrates expression of smooth muscle specific -actin and G protein-coupled receptor kinase 2 (GRK2). Passage 3 cells were grown on cover slips, washed in tris-buffered saline (TBS), and treated in 10% antigen retrieval solution (CITRA; BioGenex, San Ramon, CA). Following protein blocking with 0.3% bovine serum albumin diluted in TBS for 1 h, sections were incubated overnight at 4C with primary antibodies: (A) smooth muscle specific -actin (cbl-171, 1:2000, Millipore, Billerica, MA), (B) GRK2, (sc-562; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), or (not shown) von Willebrand factor VIII (ab7356, 1:2500, Millipore) at 4C. Endogenous peroxidase activity was blocked with 3% H2O2 solution in methanol.

Primary antibodies were localized using the V ectastain ABC-Elite peroxidase detection system (V ector Laboratories, Burlingame, CA). This was followed by reaction with diaminobenzidine as chromogen, counterstaining with hematoxylin, and visualization under phasecontrast microscopy. The observed characteristic striated pattern of localization of -actin (and lack of positive staining for von Willebrand factor VIII) confirms our cultures were predominantly VSM in the contractile state (A). In addition, the diffuse cytoplasmic localization of GRK2 demonstrates the presence of this important kinase that desensitizes the -AR upon receptor activation and is known to be highly expressed in cardiovascular tissues (B).

diffuse cytoplasmic localization of GRK2. This protein is highly expressed in cardiovascular tissues and desensitizes the -AR upon receptor activation (Pitcher et al. 1998). We have particular interest in this protein because we have previously found that measures of GRK activity and expression increase with advancing age in vascular tissue (Schutzer et al. 2001). In terms of evaluating clonal response to -AR activation, passage 3 clones (n =5 each derived from n =7 animals from each of four age groups) were analyzed for cAMP accumulation for the basal, ISO-, and FSK-stimulated treatment. Figure 2 shows the average cAMP production using our first strategy of maintaining the donor animal age for each clone. Passage 3 (Fig. 2A) clonal cell lines were initially appraised because this was the first opportunity where enough cells were available for evaluation. Both ISO- and FSK-mediated cAMP accumulation was significantly higher than basal. There was a significant age-related reduction in ISO-mediated cAMP accumulation. However, no age-related differences were found with FSK treatment. Therefore, these passage 3 cells appeared to mirror the age-related changes observed in intact vascular tissue. These passage 3 clonal populations were then passaged out to passage 6 (Fig. 2B) in hopes that the aging phenotype would be maintained. Unfortunately, the age-related decline in ISO-mediated cAMP accumulation was no longer observed at passage 6. Although the aging

phenotype was lost by passage 6, our results demonstrated that passage 3 clonal populations did mirror the response reported in the literature for intact tissue. In an alternative attempt to maintain the fidelity of aging phenotype, we employed our second strategy of segregating individual passage 3 clones into specific age categories (young, mature, old, and old-old categories), regardless of their donor animals age. We decided to ignore the donor animals age due to the documented heterogeneity of aortic vascular smooth muscle cells (Hall et al. 1991); this concept is further discussed following. Prior to age segregation, we evaluated the cAMP response and discarded clones based upon the expected response in comparison to intact tissue. The initial selection was based on basal cAMP accumulation: with the high and low 10% responders being discarded. Following, clonal lines were evaluated based on FSK-stimulated (concentration of 10 M) cAMP accumulation. Those that did not respond by fivefold and those that exceeded 50-fold over basal were also discarded. Finally, the remaining clonal lines were evaluated based on their ISO-stimulated (concentration of 10 M) cAMP accumulation. Any lines where the accumulation of cAMP to 10 M ISO stimulation exceeded the accumulation of cAMP to 10 M FSK were discarded. Also, cell lines that did not respond by a 1.5-fold increase over basal were discarded. We then segregated clones into age groups by using a four-by-two matrix where young,

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PASSAGE 3

PASSAGE 6

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cAMP/mg protein/10 min.
1600 1400 1200 1000 800 600 400 80 40 0 Basal ISO

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cAMP/mg protein/10 min. 2M 6M 12M 24M
1600 1400 1200 1000 800 600 400 80 40 0

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ISO

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Figure 2. Accumulation of cAMP in passage 3 and passage 6 clonal cells. Clonal cells derived from aortae of 2-, 6-, 12-, and 24-m-old (2, 6, 12, and 24 M, respectively) Fischer 344 rats (as described) were grown to 80% confluence in 24-well tissue culture plates at passage 3 (A) and passage 6 (B), and placed in serum-free media 24 h prior to assay. Following, cells were incubated with 25 mM isobutylmethylxanthine (Sigma, St. Louis, MO) for 20 min. cAMP accumulation was measured after exposure to 10 M isoproterenol (ISO) or forskolin (FSK; both Sigma) in duplicate wells for 10 min. Immediately after treatment, ice cold 0.1 N HCl was placed into the wells and the cells were scraped from the plate and homoginized. These homogenates were centrifuged at 500g for 15 min at 4C and the resulting cleared supernatant was analyzed with enzyme immunoassay for cAMP

content (Assay Designs, Ann Arbor, MI). The protein concentration of the pellet was determined using the BCA method (Pierce Chemical Co., Rockford, IL). Concentration of cAMP is expressed as pmol cAMP/mg protein/10 min. Shown for each passage are meanSEM for cAMP analyses each performed from n =5 clones from each of n = 7 animals from each of four age groups with basal, ISO-, and FSKstimulated treatments. *Between group significantly different from basal stimulation (p <0.05) as determined by one-way ANOV A (GraphPad Prism, San Diego, CA). Within group significantly different than 2 M; b within group significantly different than 6 and 12 M as determined by two-way ANOV A (age by treatment) with Bonferronis post hoc analysis.

mature, old, and old-old categories were defined by a fold over basal response to ISO respectively for each age designation of: greater than 12-, 812-, 48-, and 1.54-fold. Furthermore, a FSK criteria of percent response to the ISO effect of greater than 90%, 7090%, 4070%, and 1040%, respectively, for each age designation was also used to complete segregation. Using these parameters, we were able to identify three candidate lines each that matched our four age categories. These are shown in Fig. 3A. With phenotypic segregation, the age-related change in ISO-mediated cAMP accumulation was significantly reduced. As expected, this change was enhanced compared to that shown in Fig. 2A. Interestingly, the age of the donor did not necessarily confer age group segregation. That is, the young group consisted of one clone each from 2-, 12-, and 24-month-old donors; similarly, the mature group consisted of one clone each from other 2-, 12-, and 24-month-old donors; the old group consisted of one clone from a 2-month, and one clone each from a 12-, and 24-month-old donor; and finally, the old-old group consisted of one clone each from 6-, 12-, and 24-month-old donors. Therefore, donor age did not strongly correspond to the age category assigned. One possible explanation for this is that, because we used clonal selection, each clonal population was derived from a single founder cell, and each founder was derived from aortic medial explants that were made up of a heterogeneous group of cells. Therefore, we expected that the predominant

cell isolated from the aorta of a 2-month-old animal would respond fully to -AR stimulation. However, because of the overall heterogeneity, it is possible that we isolated a founder cell that did not. This holds true for the opposite situation as well: we expected that the predominant cell isolated from the aorta of a 24-month-old animal would not respond well to -AR stimulation. However, because of the overall heterogeneity, it is possible that we isolated a founder cell that did. Heterogeneity of cell properties of VSM layer of aorta is well documented. Phenotypically distinct SMC subpopulations that differed in terms of structure (Absher et al. 1989), biochemical function and growth, and even gene expression (Frid et al. 1997) have been isolated. Evidence of aortic VSMC heterogeneity related to G protein-coupled receptor function comes from Hall et al. (1991). They used a similar clonal selection strategy to this present study and established clonal VSMC lines from aorta of spontaneously hypertensive rats (SHR) and normotensive (WKY) controls. When different clonal cell lines derived from a single SHR donor animal were examined, a large range in angiotensin II receptor density values was found: this distribution covered a 73-fold range. Clonal cell lines derived from a single WKY donor also exhibited phenotypic heterogeneity. However, when the mean of all SHR clones was compared to the mean of all WKY clones, angiotensin II density was higher in the SHR strain, as predicted by physiological data. Therefore, within a single VSM layer of a single intact aorta, there are cells of

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A
cAMP/mg protein/10 min.
1500 1250 1000 750 500 250 0 Basal

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cAMP/mg protein/10 min.
1500 1250 1000 750 500 250 0 FSK Basal

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Young Mature Old Old-Old

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Figure 3. Segregation of cAMP values into age-categories. Passage 3 (A) cAMP values were segregated into specific age-categories (young, mature, old, and old-old categories), regardless of their donor animals age as described. The subsequent passage 6 response is shown in (B). *Between group significantly different from basal stimulation (p <

0.05) as determined by one-way ANOV A (GraphPad Prism, San Diego, CA). within group significantly different than young; a within group significantly different than mature, b within group significantly different than mature and old as determined by two-way ANOV A (age by treatment) with Bonferronis post hoc analysis.

multiple phenotypes and thus it is likely to be essentially random as to which subtype of cell is selected to serve as a specific clones founder. Following segregation of these 12 clonal lines (three clones each representative of four specific age groups) each derived from a single founder cell, we evaluated the subsequent passage 6 cAMP data in hopes that they would maintain the same, or at least similar characteristics in reference to adrenergic signaling as compared to passage 3. As shown in Fig. 3B, this was not the case. The apparent decline in ISO-mediated cAMP accumulation observed in passage 3 clones disappeared by passage 6. In conclusion, we sought to develop a stable vascular smooth muscle cell culture model that replicated the molecular and biochemical changes observed in blood vessels with advancing age. There is a clear need for such cells because molecular-based studies are needed to determine the mechanism of the clinical finding that blood vessels lose their ability to relax to -AR stimulation with advancing age. Cardiovascular disease is very common in the elderly. One well-documented condition is the loss of vessel relaxation caused by adrenergic signals in smooth muscle cells. Decreased relaxation contributes to alterations in blood pressure homeostasis, insufficient blood flow to organs and peripheral tissues, and atherosclerosis. We chose to use a clonal selection strategy to establish these cultures as other investigators have been successful using this technique: our experimental strategy was modeled after that published by Rothman et al. (1992). Their work produced a number of cloned cell lines, each representing a range of phenotypes. One line (named PAC1 cells) provided a standardized and homogenous population of cells with a phenotype reflective of what is observed in intact tissue for pulmonary hypertension. The cells could be passaged numerous times and maintained their phenotype in terms of response to various agonists, such

as angiotensin II, and norepinephrine. Also of importance was that these cells were usable for experimental manipulation, such as transfection of cDNA. Furthermore, because these cultured cells were derived by a clonal selection strategy, they avoided the need for mutagenesis or transformation. Others (Hall et al. 1991) also used similar techniques to this study and produce stable vascular clonal lines from SHR and WKY rats that maintained their phenotypes of interest through multiple passages. Further supporting our rationale were the findings of Bochaton-Piallat et al. (1993) who produced phenotypically distinct vascular clonal cell lines (founder cells were yielded through dilution, versus cloning rings) from different ages of Wister rats. To that end, our initial results were promising: we identified age-unique clonal cell cultures that responded to adrenergic signaling in a similar way to age-matched intact tissue. Unfortunately, this age characteristic was not maintained with passage. One factor that might have increased the likelihood of identifying age-specific clonal cell lines would be to evaluate dose and time responses for ISO and FSK. Perhaps stimulation with 10 M ISO and FSK (and incubation for 10 min) were too high and differences between clones were masked. We chose these values as these are what are commonly used when stimulating aortic smooth muscle tissue and what we and others have used in previous experiments (Deisher et al. 1989; Chin et al. 1996; Mader and Alley 1998). Similarly, we included 25 mM isobutylmethylxanthine (a phosphodiesterase inhibitor) with our cAMP assays. Again, this is typical evaluating aortic smooth muscle cAMP responses but could have masked differences between clones. We were aware that there was precedence for the possibility of not identifying age-specific clonal cell lines with passage: Crass et al. (1992) reported that cultured vascular smooth muscle cells lose the aging phenotype with

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passage. However, we attempted to mitigate this issue by using presumably pure clonal cell lines isolated from an individual founding cell, whereas Crass et al. used heterogeneous populations for their cultures. Possible explanations for why clonal selection was not successful in maintaining the aging, adrenergic phenotype with passage is unclear, especially considering the previously discussed success of this technique in establishing other stable cell lines.
Acknowledgments This work was supported by the Research Service, Department of V eterans Affairs, and the Northwest Health Foundation.

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