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J Am Soc Nephrol 15: 3016-3025, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000145529.19135.EF
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BASIC SCIENCE

Role of Parathyroid Hormone–Related Protein in the Regulation of Stretch-Induced Renal Vascular Smooth Muscle Cell Proliferation

Eric Schordan, Sandra Welsch, Sylvie Rothhut, Alain Lambert, Mariette Barthelmebs, Jean-Jacques Helwig and Thierry Massfelder

Section of Renovascular Pharmacology and Physiology (INSERM-ULP), University Louis Pasteur School of Medicine, Strasbourg, France

Correspondence to Dr. Thierry Massfelder, Pharmacologie et Physiologie Rénovasculaires (Equipe Mixte INSERM-ULP 0015), 11 rue Humann, Bâtiment 4, 1er étage, F67085 Strasbourg Cedex. Phone: 333-90-24-34-56; Fax: 333-90-24-34-59; E-mail: thierry.massfelder{at}medecine.u-strasbg.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch as a result of the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathologic conditions and contributes to vascular remodeling by modulating VSMC proliferation and death. Parathyroid hormone–related protein (PTHrP) is a locally produced poly-protein that regulates cell growth. It was shown previously that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway by interacting with its receptor, the PTH1R, but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. In the current study, VSMC that were isolated from both resistance and compliance vessels were used to study the role of PTHrP in VSMC proliferation under experimental stretch. It is shown that PTHrP gene expression is upregulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, it is demonstrated that PTHrP expression is controlled at the post-transcriptional level by stretch. Taken together, these results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Thus, in conditions in which stretch is increased, such as in hypertension or in restenosis after angioplasty, PTHrP may contribute to vessel hyperplasia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Blood vessels in vivo are continuously exposed to hemodynamic forces, including shear stress on the luminal surface generated by blood flow, cyclic distension of the vascular wall, i.e., mechanical stretch, caused by the pulsatility of the blood flow, and endocrine and local factors (e.g., angiotensin II and endothelin-1) (1). In static culture in vitro, vascular smooth muscle cells (VSMC) adopt a proliferative and dedifferentiated phenotype (1). There are conflicting data about whether stretch promotes (2–5) or inhibits (3,6,7) VSMC proliferation, and this discrepancy has been attributed to differences in VSMC phenotype. Studies using VSMC cultured under either static or stretch conditions have attempted to identify the mechanisms underlying the structural alterations observed in vivo. Several regulatory pathways have been reported, including ion channels; cell–matrix integrin interaction; myosin isoform expression; activation of tyrosine kinases and mitogen-activated protein kinase family; and autocrine production and secretion of mitogenic factors such as angiotensin II, EGF, and IGF-1 or anti-mitogenic factors such as nitric oxide (3–8).

Parathyroid hormone–related protein (PTHrP) acts, among other properties, as a local regulator of cell proliferation, differentiation, and death in normal and deceased cells and tissues (9,10). PTHrP and its receptor, the PTH/PTHrP receptor or PTH-1 receptor (PTH1R), are expressed throughout the cardiovascular system, including the intrarenal arterial tree, in VSMC and endothelial cells (9). PTHrP expression is rapidly induced in VSMC by vasoconstrictors, growth factors, and mechanical stretch, analogous to the rapid responses of cytokine mRNA (9). PTHrP expression is also stimulated in VSMC by pathologic stimuli, including atherosclerosis, restenosis after angioplasty, and hypertension (11). PTHrP is a potent relaxant factor in smooth muscle of vascular and nonvascular origins in vitro and in vivo (10). We and others have shown that the kidney vasculature appears as a privileged target for the relaxant effect of PTHrP (9–11). In addition to its relaxant effects, we and others have shown that PTHrP inhibits VSMC proliferation through its interaction with the PTH1R and stimulation of the cAMP/protein kinase A pathway resulting in cell-cycle blockade at mid-G1 (12,13). More recently, we have shown that PTHrP may also exert proliferative effects on VSMC by translocating into the nucleus through its bipartite nuclear/nucleolar localization signal (bNLS) present in the its 88 to 106 region (13). Although nuclear targets for PTHrP have not been identified, recent findings show that sequences carboxyterminal to the bNLS are necessary for the proliferative effect of PTHrP in VSMC (14). It is interesting that in VSMC that are isolated from preglomerular vessels of genetic hypertensive rats, the paradoxic effect of PTHrP on VSMC proliferation was reversed, i.e., nuclear translocation of PTHrP led to inhibition of VSMC proliferation (15). This was of course an important observation because PTHrP is overexpressed in renovascular SMC and preglomerular vessels are known to contribute greatly to the high BP in this disease. Although no clear explanation exists to explain such effect, all of the data accumulated to date thus argue for a role for PTHrP in regulating BP and arterial wall modeling and remodeling.

Previous studies in vitro and in vivo have demonstrated that stretch increases PTHrP expression in vascular and nonvascular tissues (16–19). In aortic VSMC, stretch, either alone or in combination with angiotensin II, results in a marked increase in the PTHrP mRNA level in a manner dependent on the strength of stretch (16,17). A stimulatory effect of stretch on PTHrP expression in VSMC has also been observed in various vascular disorders, including hypertension and atherosclerosis (20,21). These observations suggest that PTHrP acts as a compliance factor in these conditions. However, the possibility that PTHrP may also regulate VSMC proliferation under conditions of stretch has not been addressed. Because the kidney plays a major role in the development and maintenance of high BP in the hypertensive state, we used VSMC that were isolated from intrarenal arteries (resistance vessels) and compared their behavior with VSMC that were isolated from the aorta (compliance vessel). We now demonstrate that stretch induces PTHrP expression and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, we show that stretch regulates PTHrP expression at the post-transcriptional level by increasing PTHrP mRNA stability. Thus, under conditions in which stretch is increased, such as hypertension or restenosis after angioplasty, the resultant overexpression of PTHrP may contribute to vessel wall hyperplasia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human (h)PTHrP(1-36) and (Asn10, Leu11, D-Trp12)hPTHrP(7-34) amide were obtained from Bachem (Voisins-Le-Bretonneux, France). Polyclonal rabbit anti-PTHrP(34-53) antibody Ab2 and PTHrP(34-53) were from Calbiochem (France Biochem, Meudon, France). Affinity-purified polyclonal rabbit anti-rat (r)PTH1R antibody (peptide IV) was from Eurogentec (Angers, France). All chemicals were of the best commercial grade available.

Animals
Animals were obtained from Charles River Laboratories (L’Arbresle, France). All animal studies were performed in compliance with guidelines of the European Community and the French Government. Twelve-week-old male Wistar rats that weighed 280 to 340 g and had free access to standard food and water were anesthetized with ether and decapitated.

Isolation of Intrarenal Arteries and Culture of Renovascular SMC
Kidneys were removed, rinsed, and placed in ice-cold PBS. Renal arterial trees that contain mainly arcuate and interlobular arteries were isolated by the explant method followed by sievings, exactly as we detailed previously (22). Eight vascular trees were prepared independently from eight rats. Renovascular SMC (RvSMC) were cultured, unless otherwise specified, in DMEM supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37°C in 10% CO2. RvSMC were used at passages 6 to 20 (22).

Isolation of Aorta and Culture of Aortic SMC
Aortic VSMC (AoSMC) were obtained by an explant technique, as detailed previously (22). Briefly, the thoracic aorta was thoroughly dissected free from connective tissue and cut longitudinally. Intimal and adventitial layers were removed, and the aorta was cut into small pieces. Eight aortic beds were prepared independently from eight rats. Aortic explants were processed as renovascular explants and AoSMC cultured as RvSMC.

Plasmids and Stable Transfection of VSMC
Cells that were derived from one representative RvSMC and AoSMC cell line were used (see the Results section). For overexpressing PTHrP or inhibit endogenous rPTHrP expression, cells were transfected with a pcDNA3 vector (Invitrogen, Cergy-Pontoise, France) construct in which hPTHrP(1-139) cDNA had been subcloned in either a sense (pcDNA3-PTHrP) or an antisense orientation (pcDNA3-AS:PTHrP). Cells were also transfected with a pcDNA3-PTHrP construct in which the bNLS of PTHrP, i.e., amino acids 88 to 92 and 102 to 106, was deleted by directed mutagenesis (pcDNA3-{Delta}NLS:PTHrP) (13). Cells that were transfected with the empty vector pcDNA3 served as controls (pcDNA3). Transfection was carried out according to the manufacturer’s protocol. Transfected cells were selected using G418 at 75 µg/ml.

Mechanical Cyclic Stretch
Untransfected and transfected RvSMC and AoSMC cells were seeded at a density of 4000 cells/cm2 onto Flex culture plates (Flexcell Corporation, McKeesport, PA) coated with rat collagen I (Sigma, St. Quentin Fallavier, France). Cells were grown for an initial 48 h to allow cell attachment and spreading. Culture medium was replaced by DMEM supplemented with 0.1% BSA (serum-free medium) for a second 48h period to induce quiescence. Culture medium was then replaced by fresh medium supplemented with 10% FBS or 0.1% BSA. Cells were exposed for 4 h to 7 d to an equiaxial mechanical cyclic stretch of 10% magnitude and a frequency of 1 Hz using a homemade device. RvSMC and AoSMC cells were also grown on Flex plates under identical conditions but were not exposed to stretch (static control).

Cell Growth
RvSMC and AoSMC cells were seeded on six-well plates at a density of 4000 cells/cm2 and grown in serum-containing medium. For determining the population doubling time (PDT), i.e., cell cycle duration at exponential growth (typically between days 2 and 6), adherent cells were counted manually for 14 d using a hemocytometer at 2d intervals. For evaluating the effect of stretch on cell growth, untransfected or transfected RvSMC and AoSMC were processed as indicated in the stretch protocol. Adherent cells were counted at each time point using a hematocytometer.

RNA Extraction and Semiquantitative Reverse Transcriptase–PCR
Total RNA was extracted from untransfected or transfected RvSMC and AoSMC cells that were grown under static or stretched conditions using the TRIzol method (Invitrogen, Cergy-Pontoise, France) according to the manufacturer’s protocol. The relative abundance of rPTHrP, hPTHrP, and rPTH1R was analyzed by reverse transcriptase–PCR (RT-PCR) using rat glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) expression to normalize, exactly as described before (15). Primers for rPTHrP, hPTHrP, rPTH1R, and rGAPDH have been described previously (23). PCR products were identified on agarose gels by their expected size of 320 bp (rPTHrP), 535 bp (hPTHrP), 508 bp ({Delta}NLS-hPTHrP), 817 bp (rPTH1R), and 415 bp (rGAPDH). Control reactions were performed by omitting reverse transcriptase. Band intensities were quantified by a gel analysis software (Sigma Gel; Jandel Scientific, Erkrath, Germany).

Quantitative Real-Time PCR Analysis
rPTHrP cDNA was amplified using the LightCycler-FastStart DNA Master SYBR Green kit (Roche Diagnostics, Meylan, France). In addition, a standard curve was obtained for rGAPDH by serial dilutions of mixed samples cDNA. A 20-µl mix contained 4 mM MgCl2, 0.5 µM each primer set, 0.2 µM SYBR Green probe, 0.4 µM hybridization probe, 1x FastStart reaction buffer (DNA polymerase, dNTP, buffer), and 2 µl of cDNA (50 ng). PCR reactions for rPTHrP and rGAPDH were as follows: 95°C for 10 min followed by 40 cycles at 95°C for 10 s, 60°C for 5 s, and 72°C for 12 s (rPTHrP) or 16 s (rGAPDH). cDNA was replaced by PCR-grade water as a negative control. Each sample was run twice and quantified with the LightCycler analysis software according to the manufacturer’s protocol (Roche Diagnostics). Relative expression of rPTHrP in samples was calculated by quantifying rPTHrP level normalized to rGAPDH, and results were expressed as a percentage of the expression obtained at 0 h set to 100%.

PTH1R Western Blot Analysis
PTH1R expression in RvSMC and AoSMC cells was analyzed essentially as described (23) on 30 µg of protein extract, using an anti-PTH1R antibody (Eurogentec). A polyclonal mouse anti–{beta}-actin antibody (Sigma) was used for visualization of protein gel loading.

Immunofluorescence
RvSMC and AoSMC cells were grown in glass chamber slides for 48 h and processed, as detailed (13). Cells were incubated with the polyclonal anti-PTHrP Ab2 antibody at 5 µg/ml at room temperature for 2 h. A FITC-conjugated anti-rabbit secondary antibody was used for detection of the PTHrP. After final washes, cells were mounted in antifading solution (DakoCytomation, Trappes, France) and visualized with a Nikon (Tokyo, Japan) inverted fluorescence microscope. As a competition control, the primary antibody was preincubated overnight at 4°C with 10–6 M PTHrP(34-53) peptide. As an additional control, nonimmune rabbit IgG (Sigma) was used instead of primary antibody.

PTHrP Promoter Activity Measurement
RvSMC and AoSMC cells were seeded at 4000 cells/cm2 in Flex plates and grown for 48 h in serum-containing medium. Culture medium was replaced by serum-free medium for 48 h to induce quiescence. Cells then were transiently transfected with a construct that contained the 5'-flanking promoter regions (promoters P1, P2, and P3) of the hPTHrP gene ligated to a promoterless bacterial chloramphenicol acetyltransferase (CAT) gene (24). Transfection was carried out with 1 µg/cm2 of the PTHrP promoter CAT plasmid or the promoterless CAT plasmid, using the Lipofectamine reagent. RvSMC and AoSMC cells were then grown for 48 h in serum-free medium under static or stretched conditions and analyzed for CAT enzyme expression using the CAT enzyme ELISA kit (Roche Diagnostics). Results were normalized and expressed as pg CAT enzyme/mg protein.

Actinomycin D Experiments
RvSMC and AoSMC cells were plated as above. Cells were then grown for 0 to 8 h in serum-free medium under static or stretched conditions in the presence or absence of 5 µg/ml actinomycin D (Sigma). Total RNA was isolated at each time point, and PTHrP expression was analyzed by RT-PCR using rGAPDH for normalization. The relative intensity of the PTHrP mRNA signal (rPTHrP/rGAPDH ratio) in each lane was then expressed as a percentage of the signal obtained at the time of actinomycin D addition (0 h) set to 100%.

Statistical Analyses
All values are expressed as mean ± SEM. Values were compared using multifactorial ANOVA followed by the Student-Newman-Keul’s test for multiple comparisons. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth Characteristic of RvSMC and AoSMC
Growth of RvSMC and AoSMC expressed as PDT were studied on eight independent rat vascular explants. RvSMC grew more slowly than AoSMC (41.5 ± 4 h in RvSMC versus 24.3 ± 1.3 h in AoSMC, n = 7; P < 0.01), as previously observed (22). Subsequent experiments were performed on RvSMC and AoSMC derived from one representative explant, exhibiting a PDT value comparable to the overall mean value (42 h for RvSMC and 23.7 h for AoSMC; Figure 1).



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  		Figure 1. Population doubling time (PDT) of smooth muscle cells that were isolated from renal arterial trees (RvSMC) and aorta (AoSMC) of Wistar rats. {blacksquare}, mean PDT value of RvSMC and AoSMC cell lines; {square}, PDT value of the RvSMC and AoSMC cell lines that were selected for subsequent analysis. *P < 0.05, RvSMC versus AoSMC; n = 8.

 
Mechanical Cyclic Stretch Inhibits RvSMC and AoSMC Proliferation
In serum-containing medium, proliferation of SMC from both origins was significantly decreased by stretch (Figure 2). Two effects were observed: First, a 2d delay in cell proliferation and, second, a significant lower cell density at plateau phase (Figure 2). In serum-free medium, cells did not proliferate regardless of vessel origin or stretch status.



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  		Figure 2. Effect of 10% mechanical stretch on RvSMC (A) and AoSMC (B) proliferation. Cells were seeded at 4000 cells/cm2 onto Flex culture plates and submitted to a 10% elongation in either the presence (10% FBS) or the absence (0.1% BSA) of serum after a 48-h period of quiescence. Adherent cells were counted at the indicated time points. In the absence of serum, cells were not proliferating. In the presence of serum, stretch induced an inhibitory effect on cell proliferation regardless of SMC origins. Note the lower plateau phase obtained in cells that were submitted to stretch. Results are presented as mean ± SEM (n = 3 in the absence of serum and n = 10 in the presence of 10% FBS). *P < 0.01 stretch versus static cells.

 
Mechanical Cyclic Stretch Stimulates PTHrP Expression in RvSMC and AoSMC
In serum-containing medium, induction of PTHrP expression by stretch was evident in RvSMC beginning at 24 h and was maintained for at least 96 h (Figure 3A). We confirmed the stimulatory effect of stretch on PTHrP expression for the 24 h time point at the mRNA level by quantitative analysis (Figure 3B) and at the protein level by immunofluorescence (Figure 3C). It is interesting that in serum-free medium, a significant increase in PTHrP expression was also observed after 24 h of stretch at both mRNA (Figure 3A) and protein (data not shown) levels. In contrast, PTH1R expression was decreased by stretch at both mRNA (Figure 3D) and protein (Figure 3E) levels at all time points, reflecting probably an inhibitory effect of upregulated PTHrP on PTH1R expression. Similar results were obtained in AoSMC cells (data not shown).



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  		Figure 3. Effect of 10% mechanical stretch on parathyroid hormone–related protein (PTHrP) and rPTH1 expression in vascular smooth muscle cells (VSMC). Semiquantitative reverse transcriptase–PCR (RT-PCR) analysis of rPTHrP (320 bp) in RvSMC that were grown under stretched or static conditions at the various time points indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 415 bp) expression was used as the housekeeping gene for semiquantification of PTHrP expression. Shown is a representative ethidium bromide–stained agarose gel from at least three experiments. Stretch increases PTHrP expression at all time points tested. It is interesting that in the absence of serum (0.1% BSA), PTHrP expression was also increased by stretch after 24 h. (B) Bars represent a real-time PCR quantification of the PTHrP transcript in RvSMC at 0 and 24 h grown under static (0 and 24) or stretch (24S) conditions in the presence of serum. Results are presented as mean ± SEM (n = 3 to 4). *P < 0.05 in 24S versus 0 and 24. (C) Immunofluorescence micrographs showing PTHrP upregulation in RvSMC in response to stretch. RvSMC were grown for 24 h under either static (upper two micrographs) or stretched (lower two micrographs) conditions and immunostained for PTHrP. Results confirmed the stimulatory effect of stretch on PTHrP expression at the protein level. (D) RT-PCR analysis of PTH1R (817 bp) and GAPDH expression in RvSMC that were grown under stretched or static conditions at the various time points indicated. In contrast to PTHrP, PTH1R expression was decreased by stretch. Shown is a representative agarose gel from at least three experiments. (E) A typical immunoblot of the PTH1R protein in RvSMC that were grown for 24 h under either static or stretch conditions showing the stretch-induced decrease in PTH1R expression. Shown is a representative immunoblot from four experiments.

 
PTHrP Opposes the Inhibitory Effect Induced by Mechanical Cyclic Stretch on RvSMC and AoSMC Proliferation
As shown in Figure 4A, transfections with pcDNA3-PTHrP and pcDNA3-AS:PTHrP constructs were highly efficient in increasing or inhibiting PTHrP expression, respectively, consistent with previous results obtained in VSMC (13). Decreasing PTHrP expression with the AS:PTHrP construct did not affect RvSMC growth under static or stretched conditions (Figure 4B). However, in RvSMC that were transfected with the sense PTHrP construct, while the delay in cell proliferation was maintained in stretched cells, the plateau phase was no longer different between cells that were grown in static versus stretch conditions (Figure 4C). That no effect was observed in RvSMC that were transfected with the AS:PTHrP construct on cell proliferation under stretch condition therefor ise unexpected but, as shown in Figure 4D, may be related to the incomplete effectiveness of the AS:PTHrP transfection to decrease stretch-induced PTHrP upregulation. Similar data were obtained in AoSMC (data not shown).



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  		Figure 4. Stretch-induced PTHrP opposes the inhibitory effect of stretch on VSMC proliferation. (A) RT-PCR analysis of rPTHrP (left; 320 bp), hPTHrP (right; 535 bp), and GAPDH expression in RvSMC that were transfected with the PTHrP antisense construct (pcDNA3-AS:PTHrP; AS) or the PTHrP sense construct (pcDNA3-PTHrP; PrP) constructs or pcDNA3 alone (V). Shown are representative ethidium bromide–stained agarose gels from at least three experiments. (B) The graph shows proliferation of vector (pcDNA3) or PTHrP antisense construct (pcDNA3-AS:PTHrP)-transfected RvSMC that were grown in static or stretch condition during the indicated time. Transfection with the AS construct did not affect cell proliferation whether they were stretched or not. For clarity, results are presented as mean ± SEM (n = 5) calculated from the cell density obtained after 5 d in static condition that was set to 100%. *P < 0.01 stretch versus static RvSMC. (C) The graph shows proliferation of vector (pcDNA3) and PTHrP sense construct (pcDNA3-AS:PTHrP)-transfected RvSMC that were grown in static or stretch condition during the indicated time. As in B, results are presented as mean ± SEM (n = 5) calculated from the cell density obtained after 5 d in static condition that was set to 100%. In RvSMC that were transfected with PTHrP, proliferation of stretched cells was no longer inhibited. (D) RT-PCR analysis of rPTHrP and GAPDH expression in RvSMC that were transfected with the PTHrP antisense construct (AS) or the vector alone (V) submitted to a 24-h stretch period. AS 0 represents level of rPTHrP expression in cells that were transfected with the PTHrP antisense construct grown under static condition. Under stretch, the PTHrP antisense construct was less efficient in inhibiting PTHrP expression, which went up to 50% of the level seen in vector-transfected RvSMC. Shown is a representative agarose gel from at least three experiments.

 
PTHrP Acts through on Intracrine Pathway to Oppose the Inhibitory Effect Induced by Mechanical Cyclic Stretch on VSMC Proliferation
PTHrP was detected in the nucleus of PTHrP-transfected RvSMC (Figure 5A). PTHrP was also present in nontransfected cells and in cells that were transfected with the vector alone or with the pcDNA3-{Delta}NLS:PTHrP construct, although in a lower percentage of cells (data not shown), consistent with previous findings in VSMC (13). As shown in Figure 5B, transfection with the pcDNA3-{Delta}NLS:PTHrP construct was highly efficient but did not affect RvSMC growth under static or stretched conditions (Figure 5C). Furthermore, the continuous exposure of untransfected RvSMC to a high dose of either PTHrP(1-36) (Figure 5D) or (Asn10, Leu11, D-Trp12)hPTHrP(7-34) amide (Figure 5E), a potent RPTH1 antagonist, did not modify their proliferation, whether they were stretched or not. Similar data were obtained in AoSMC (data not shown).



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  		Figure 5. PTHP acts through the intracrine pathway to oppose the inhibitory effect of stretch on VSMC proliferation. (A) Immunofluorescent staining of RvSMC that were transfected with the PTHrP sense construct (pcDNA3-PTHrP) showing cytoplasmic and nuclear staining of PTHrP. (B) RT-PCR analysis of hPTHrP expression and GAPDH in nontransfected RvSMC (Ctl) and in RvSMC that were transfected with the PTHrP sense construct (pcDNA3-PTHrP; PrP; 535 bp) or the NLS-deleted PTHrP construct (pcDNA3-{Delta}NLS:PTHrP; {Delta}NLS; 508 bp) or pcDNA3 alone (V). Shown is a representative ethidium bromide–stained agarose gel from at least three experiments. (C) The graph shows proliferation of vector (pcDNA3) and NLS-deleted PTHrP construct (pcDNA3-{Delta}NLS:PTHrP)-transfected RvSMC that were grown in static or stretch condition during the indicated time. Results are presented as mean ± SEM (n = 5 to 6) calculated from the cell density obtained after 5 d in static condition that was set to 100%. Transfection with this construct did not affect cell proliferation whether they were stretched or not. (D and E) RvSMC were continuously exposed to exogenous PTHrP(1-36) at 10–7 M (D) or (Asn10, Leu11, D-Trp12)hPTHrP(7-34) amide (E) and grown under static or stretch condition, and cell density was measured at the indicated time points. The continuous presence of high dose of the PTH1R agonist or antagonist did not affect cell proliferation whether they were stretched or not. Results are presented as mean ± SEM (n = 3 to 10) calculated from the cell density obtained after 4 d in static condition that was set to 100%. *P < 0.01 stretch versus static RvSMC. Magnification, x400 in A.

 
Mechanical Cyclic Stretch Stimulates PTHrP Expression through mRNA Stabilization
Further studies were performed to gain insight into the mechanism accounting for stretch-induced PTHrP expression. Because angiotensin II has been shown first to control PTHrP expression (25) in VSMC and second to be induced in VSMC submitted to an experimental stretch (26,27), we first tested the hypothesis that angiotensin II is involved in stretch-induced PTHrP upregulation. The PTHrP expression profile in response to stretch remained unchanged in the presence of 10–7 M losartan, an angiotensin II receptor antagonist (Figure 6A), ruling out the involvement of angiotensin II in PTHrP expression.



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  		Figure 6. Mechanism of stretch-induced PTHrP expression. (A) RT-PCR analysis of rPTHrP and GAPDH expression in RvSMC that were grown under stretch or static condition in the presence or absence of the angiotensin II receptor antagonist losartan at the various time points indicated. PTHrP expression profile was not altered by the presence of the angiotensin II antagonist. (B) Bars represent chloramphenicol acetyltransferase (CAT) expression in RvSMC that were transiently transfected with an hPTHrP promoter CAT expression plasmid. Stretch did not alter PTHrP promoter activity. Results are presented as mean ± SEM (n = 6). (C) Increase in rPTHrP mRNA stability in RvSMC that were grown under stretch condition as compared with cells that were grown in static condition. Results are presented as mean ± SEM (n = 3). *P < 0.01 stretch versus static RvSMC. Representative agarose gels are shown at the top right for static and stretched RvSMC.

 
As shown in Figure 6B, hPTHrP transcriptional activity in RvSMC was not altered by stretch. However, analysis of the decay of the rPTHrP mRNA in RvSMC that were exposed to the transcriptional inhibitor actinomycin D revealed that PTHrP half-life is significantly increased in RvSMC in response to stretch (2.9 ± 0.3 and 6.5 ± 1.4 h in RvSMC grown in static versus stretch conditions, respectively; n = 3; *P < 0.01; Figure 6C). Similar data were obtained in AoSMC (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The luminal surface of blood vessels is lined with endothelial cells so that shear stress is predominantly sensed by and acts on endothelial cells. However, both endothelial cells and VSMC are subjected to cyclic stretch. In normal conditions, VSMC are quiescent and contractile (1). In response to pathologic stress, such as a sustained elevation in BP, VSMC develop a proliferative, hypertrophic, and secretory phenotype (1). These alterations result in vascular remodeling characterized by cellular hyperplasia, hypertrophy, apoptosis, enhanced protein synthesis, and extracellular matrix reorganization.

There are conflicting data about whether stretch inhibits or stimulates VSMC proliferation (2–7). Thus, for example, Chapman et al. (6) showed that cyclic stretch causes cell-cycle arrest at the G1/S phase transition in VSMC by increasing the expression of the cyclin-dependent kinase inhibitor p21. In contrast, Iwasaki et al. (4) demonstrated that mechanical stretch stimulates VSMC growth via the EGF receptor. In both cases, VSMC were isolated from the rat thoracic aorta, but in one study (6), cells were obtained by collagenase dispersion and in the other (4) by the explant method. Although different cell isolation methods could explain divergent results, this is not corroborated by the above-mentioned study (6) and another study (5). Another possibility is that the level of stretch applied to VSMC in vitro will direct the cell growth response to the stretch stimulus. However, opposed effects have been reported on VSMC growth in cells that were submitted to physiologic stretch (10% elongation) as well as in cells that were exposed to supraphysiologic stretch (15 to 25% elongation) (3–7,26–28). The most plausible hypothesis to explain these conflicting results is the difference in phenotype between VSMC. Such variability in VSMC phenotypes between different vessels and within the same vessel has been confirmed by many studies (29,30). In addition, the heterogeneous nature of VSMC developmental origin provides another level of complexity in VSMC growth mechanisms (30). In the current study, we used VSMC that were isolated from both resistance and compliance vessels. We show that the effect of stretch on VSMC proliferation is in fact more complex than previously reported. Indeed, VSMC respond to stretch by a first delay in cell proliferation. In our condition, this delay lasted 2 d in both RvSMC and AoSMC. Cells then proliferate, but cell density reached at plateau phase by cells that were grown under stretch condition was lower than in static condition. In static conditions, VSMC stopped growing after reaching confluence. In additional pilot experiments, we obtained similar results when cells were submitted to a supraphysiologic stretch (20% elongation; data not shown). However, because such level of stretch resulted in a large amount of cell detachment, further studies were performed at 10% elongation for which cell detachment was not observed. Because cell density was higher in cells that were grown in static condition as compared with cells that were grown under stretch condition, the lower plateau phase reached by stretched cells cannot be due to medium impoverishment or contact inhibition but most probably to a cell growth arrest appearing before confluence.

The diversity in VSMC response to stretch reflects and is reflected by the various mechanisms that have been evidenced in specific studies in vitro. From previous data, we hypothesize that PTHrP might belong to the family of growth factors that regulate stretch-dependent VSMC growth. Our findings in VSMC that were derived from both resistance and capacitance vessels confirm earlier results of a stretch-induced increase in PTHrP expression at both mRNA and protein levels in VSMC from different vessel and species (including human) origins. However, we show here that the stretch-induced PTHrP expression in VSMC is long-lasting, lasting at least 96 h. Using RvSMC and AoSMC that were stably transfected with various PTHrP constructs, our finding is that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In PTHrP-transfected cells, there was no difference in cell density at plateau phase whether they were stretched or not, suggesting that the nuclear presence of PTHrP stimulates cell-cycle progression. Because PTHrP has been hypothesized to bind homopolymeric and total cellular RNA (31), the hypothesis has emerged that PTHrP might regulate RNA metabolism and particularly gene transcription. However, such a property for PTHrP has not been confirmed. The results described here should now help not only to identify other nuclear targets for PTHrP but also to decrypt the molecular mechanism of PTHrP-induced VSMC proliferation.

In response to stretch, several intracellular signaling pathways are activated and eventually result in increased transcription of genes that harbor "stretch-response elements" in their promoters. These include transcription factors such as c-fos and c-jun, increasing the activity of the AP-1 transcription complex (2,24,32,33). As a consequence, genes that bear AP-1 binding sites in their 5' regulatory sequence may also be stimulated. Although it is actually unknown whether the PTHrP gene contains a stretch-responsive element, AP-1 and AP-2 binding sites are present in its 5' region. However, our experiments indicate that stretch does not alter PTHrP gene transcription but doubles PTHrP mRNA half-life. Further study is required to resolve the exact molecular mechanisms by which the stretch enhances

PTHrP mRNA stability.

Taken together, our studies argue for a critical role for PTHrP in the growth control processes in response to stretch in VSMC. Our results should provide new avenues in the comprehension of the molecular pathways involved in vascular wall homeostasis in normal and pathologic conditions.


   Acknowledgments
 
This work was supported by grants from the French National Institut of Health and Medical research (INSERM) and the University Louis Pasteur. This work is part of the PhD thesis of E.S., who is supported by a fellowship from the French Ministry of higher education.

D. Kuhlwein is thanked for secretarial help.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 3, 2004. Accepted for publication August 27, 2004.


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