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Endocrinology Vol. 140, No. 4 1826-1833
Copyright © 1999 by The Endocrine Society

ARTICLES

Reduced Blood Pressure and Increased Sensitivity of the Vasculature to Parathyroid Hormone-Related Protein (PTHrP) in Transgenic Mice Overexpressing the PTH/PTHrP Receptor in Vascular Smooth Muscle1

Jin Qian, John N. Lorenz, Shigeto Maeda, Roy L. Sutliff, Craig Weber, Toshiyuki Nakayama, Melissa C. Colbert, Richard J. Paul, James A. Fagin and Thomas L. Clemens

Division of Endocrinology and Metabolism, Departments of Medicine (J.Q., S.M., T.N., J.A.F., T.L.C.) and Molecular and Cellular Physiology (J.N.L., R.L.S., C.W., R.J.P., J.A.F., T.L.C.), University of Cincinnati, and the Department of Pediatrics, Children’s Hospital (M.C.C.), Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Thomas L. Clemens, Ph.D., Division of Endocrinology and Metabolism, University of Cincinnati, Room 5564, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0547. E-mail: clementl{at}uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH-related protein (PTHrP) is produced in vascular smooth muscle, where it is postulated to exert vasorelaxant properties by activation of the PTH/PTHrP type 1 receptor. As a model for studying the actions of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress the PTH/PTHrP receptor (PTHrP-R) in smooth muscle. Oocyte injection with a SMP8-PTHrP-R fusion construct yielded six founder mice. F1 offspring were viable and demonstrated selective overexpression of the SMP8-PTHP-R messenger RNA in smooth muscle-rich tissues. Baseline blood pressure measured in conscious mice by tail sphygmomanometry was significantly lower in the receptor-overexpressing mice than that in controls (117 ± 4 vs. 133 ± 3 mm Hg; P < 0.05). In anesthetized animals, iv infusion of PTHrP-(1–34)NH2 caused a significantly greater reduction in blood pressure and total peripheral resistance in transgenic mice than in control animals. Vascular contractility was studied in paired, isometrically mounted aortas from 9-week-old transgenic and wild-type mice. The force of contraction in response to phenlyephrine was not significantly different between transgenic and wild-type mice. However, PTHrP-(1–34) NH2 relaxed aortic vessel preparations from transgenic mice to a greater extent than in controls (77.1 ± 3% vs. 38.4 ± 4%; P < 0.001). To determine the impact of overexpression of PTH/PTHrP type 1 receptor and its ligand on the development of the cardiovascular system, double transgenic mice were created by crossing SMP8-PTHrP-R transgenic mice with mice overexpressing PTHrP (SMP8-PTHrP). Double transgenic mice died around day E9 with abnormalities in the developing heart. In conclusion, overexpression of PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure, probably through sustained activation of the receptor by endogenous ligand. The cardiovascular defects observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP may play a role in the normal development of the cardiovascular system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH-RELATED protein (PTHrP) is a recognized locally active autocrine/paracrine peptide that is expressed in many tissues (1). PTHrP modulates the developmental program of several cell types and also appears to play a role in the transplacental movement of calcium (1). In the adult animal, the expression of PTHrP is more restricted and is particularly abundant in smooth muscle, including uterus (2), bladder (3), stomach (4), and blood vessels (5). Although the precise function of PTHrP in smooth muscle is still unclear, marked induction by mechanical stretch (6, 7) and vasoconstrictors together with its ability to relax smooth muscle (8) suggest that this peptide acts as a local smooth muscle compliance factor to accommodate flow or response to contractile stimuli in these organs. In addition to its effects on smooth muscle contractility, PTHrP modulates the growth of vascular smooth muscle cells. Vascular smooth muscle cells exposed to N-terminal PTHrP peptides in culture undergo growth arrest (9). Interestingly, enforced overexpression of PTHrP in stably transfected vascular smooth muscle cells is associated with an increase in proliferation and nuclear accumulation of the protein (10).

PTHrP shares N-terminal sequence homology with PTH, so that both peptides activate a common G protein-linked receptor termed the PTH/PTHrP type 1 receptor. PTH produced in the parathyroid gland regulates calcium and phosphorous metabolism by activating the PTH/PTHrP receptor (PTHrP-R) in bone and kidney (11). The ability of PTHrP to activate the PTH/PTHrP type 1 receptor in these tissues accounts for its hypercalcemic actions in patients with pathological overexpression of the peptide due to unregulated production by cancer cells (12). However, in contrast to PTH, which acts as a classical endocrine hormone, PTHrP appears to exert its normal effects locally in an autocrine/paracrine fashion (1). The PTH/PTHrP type I receptor is expressed in rat vascular smooth muscle cells, and its activation by PTHrP-(1–141) and synthetic N-terminal PTHrP fragments stimulates adenylyl cyclase activity (13). However, there is evidence for additional PTH/PTHrP-R. For example, Usdin et al. (14) isolated a second PTH receptor, termed the PTH-2 receptor, which bears homology to the PTH/PTHrP type 1 receptor and other members of the secretin G protein receptor family. Studies to date in cells transfected with recombinant PTH type 2 receptors suggest that it preferentially binds PTH and is relatively unresponsive to N-terminal PTHrP fragments (14, 15). However, the PTH-2 receptor is expressed in vascular smooth muscle and endothelial cells (16). Other studies showed that a synthetic PTHrP peptide comprising amino acids 38–64, which did not activate the PTH/PTHrP type 1 receptor, stimulated a calcium transient in squamous carcinoma cells (17), suggesting the existence of a novel midregion PTHrP receptor. In addition, the recent observations (10) of nuclear localization of PTHrP in vascular smooth muscle cells suggest that PTHrP may also function intracellularly. Thus, it is likely that multiple PTH/PTHrP-R and effectors have evolved to mediate distinct functions of this protein in different cell types.

To examine the role of PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that selectively overexpressed PTHrP in vascular smooth muscle (18). These mice developed hypotension consistent with the predicted role of this protein as a local vasodilator. However, the constitutive expression of ligand in these mice led to desensitization of their vasculature to PTHrP, which compromised our ability to define the full range of activity of PTHrP in vascular smooth muscle. Therefore, in the present studies we targeted the expression of the PTH/PTHrP type I receptor to smooth muscle of transgenic mice using the SMP8 {alpha}-actin promoter. We reasoned that if the cardiovascular actions of PTHrP were mediated by the PTH/PTHrP type I receptor rather than by another receptor, then the phenotype of the SMP8-PTHrP type I receptor (SMP8-PTHrP-R)-overexpressing mice would recapitulate that seen in the SMP8-PTHrP transgenic mice. Indeed, mice overexpressing SMP8-PTHrP-R were also hypotensive compared with wild-type littermates. Interestingly, whereas the SMP8-PTHrP-R transgenic mice developed normally, double transgenic mice overexpressing both SMP8-PTHrP and SMP8-PTHrP-R died at approximately embryonic day 9.5 with severe abnormalities in the developing heart.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic mice
A 3.6-kb fragment of the SMP8-chloramphenicol transferase plasmid (19) was ligated into a plasmid encoding the rabbit ß-globin second intron and the bovine GH polyadenylase sequence to generate SMP8-BGGHpA. A 1.9-kb fragment of plasmid R15B encoding the mouse PTH/PTHrP-R (20) was excised using EcoRI and XcmI, then blunt end ligated to SMP8-BGGHpA. The SMP8-PTHrP-R fusion gene was linearized and purified before pronuclear injection at the transgenic mouse facility of the University of Cincinnati. Microinjected eggs were implanted into the oviduct of pseudopregnant female mice and carried to term. Positive founders were identified by Southern blotting and bred to wild-type FVB-N mice for propagation of the line. Heterozygotes and wild-type progeny from the F1 and subsequent generations were selected by Southern blotting of EcoRI-restricted genomic DNA. Hybridization was performed with a mouse PTHrP-R complementary DNA (cDNA) labeled by random priming (Prime-It II kit, Stratagene, La Jolla, CA).

RNA isolation and Northern blot analysis
Total RNA was isolated from tissues with RNASTAT-60 (Tel-Test, Inc., Friendswood, TX). Ten micrograms of tissue total RNA were gel-separated, transferred to Nylon membrane, and then hybridized with random primed mouse PTHrP-R cDNA. The ethidium bromide-stained 18S ribosomal RNA was used as an index of equivalency of RNA loading.

Tissue morphometry of transgenic mice
Transgenic mice and their wild-type littermates were killed by CO2 asphyxiation. After obtaining body weights, organs were dissected, tissue-blotted, and weighed. Microscopic images of aortic sections of SMP8-PTHrP-R transgenic mice and their age-matched wild-type controls were color-captured into the computer and analyzed as previously described (19).

Cardiovascular hemodynamic and vascular contractility measurements
Blood pressure measurements in conscious mice, measurement of mean arterial pressure and cardiac output in anesthetized intact mice, and vascular contractility measurements were performed exactly as described previously (18). All data were analyzed using a two-factor ANOVA, with repeated measures on the second factor. Comparisons of individual means were performed using individual contrasts.

Creation of SMP8-PTHrP/SMP8-PTHrP-R double transgenic mice
F1 progeny from SMP8-PTHrP-overexpressing mice (line 375) (18) were mated with F1 offspring from SMP8-PTHrP-R-overexpressing mice. Genotyping of live offspring and embryos was performed using PCR. Primers for the mouse PTHrP-R were: forward, 5'-CTTGAAGTCCAA TGCCAGTGTCCAG-3', corresponding to nucleotides 1460–1484 of the mouse PTHrP-R; and reverse, 5'-GACACCTACTCACACAATGC-3', corresponding to the bovine GH polyadenylase sequence. The primers for the SMP8-PTHrP were: forward, 5'-CAGAATCCTGCAATATGTCC-3'; and reverse, 5'-CTGTGTCTGAACATCAGCTC-3', corresponding to nucleotides within 1048–1442 bp in the PTHrP human cDNA (22). Embryos collected from timed pregnant females were examined as whole mounts. All animals received humane care in compliance with the local institutional animal care and use committee.

Peptides
Synthetic PTHrP-(1–34)NH2 was purchased from Bachem (Torrance, CA) and stored at -20 C in 0.01 N acetic acid. Acetylcholine and phenlyephrine were purchased from Sigma Chemical Co. (St. Louis, MO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of SMP8-PTHrP-R mice and examination of transgene expression
A total of 6 founder mice were identified from 29 screened. Four lines were propagated, and the level of transgene expression was examined in tissues by Northern blot analysis. The endogenous 2.4-kb PTHrP-R messenger RNA (mRNA) transcript was detectable in 10 µg total RNA in kidney of wild-type mice, but was undetectable in other tissues (Fig. 1Go). The transgenic SMP8-PTHrP-R mRNA was detected in smooth muscle-rich tissues and was expressed at highest levels in bladder, stomach, and colon, but was not detected in brain, liver, or thymus. Figure 1BGo shows a Northern blot subjected to phosphorimaging for an extended period to compare the relative expressions of transgenic SMP8-PTHrP-R mRNA in bladder and aorta among F1 offspring derived from the 4 individual SMP8-PTHrP-R founders. The level of transgene expression varied among lines and was highest in lines 122 and 127. These mice were propagated for further study.



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  		Figure 1. A, Northern blot analysis of SMP8-PTHrP transgene expression in tissues from a representative transgenic mouse (line 122) and a wild-type littermate. Ten micrograms of total RNA were gel separated, transferred to a nylon membrane, and then hybridized with a mouse PTH/PTHrP receptor cDNA (top panel). The SMP8-PTHrP-R transgene mRNA was expressed at high levels in smooth muscle-rich tissues. The lower panel shows the ethidium-stained gel used as an index of equivalence of RNA loading. B, SMP8-PTHrP-R mRNA transgene expression in aorta and bladder from four separate transgenic mouse lines (869, 122, 124, and 127).

 
Total body and organ weights of SMP8-PTHrP-R transgenic mice
Total body and individual organ weights were determined in F1 mice derived from line 122 at 7 weeks of age (Table 1Go). The total body weight of the transgenic mice was marginally, but significantly, lower than that of wild-type controls. The wet weight of the stomach from transgenic mice was significantly greater than that in controls after adjusting for body weight. There were no major differences in the weights of any other organs after controlling for body weight. Similar results were observed in mice from line 127 SMP8-PTHrP-R (data not shown). Histomorphometry was performed on transverse sections of aorta from transgenic and wild-type mice. Their were no significant differences in outer perimeter, luminal area, or medial area or thickness in transgenic vs. wild-type mice (data not shown).


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  		Table 1. Total body and tissue weights of SMP-PTHrP-R mice and age-matched wild-type controls

 
Effects of overexpression of SMP8-PTHrP-R on blood pressure and hemodynamics
As shown in Fig. 2Go, blood pressure measured in awake mice over a 5-day period was significantly lower in transgenic animals (line 122) than in wild-type animals (117 ± 4 vs. 133 ± 3 mm Hg; P < 0.05). Heart rate was slightly lower in transgenic mice compared with controls, but this difference did not reach significance. Analogous measurements in a second line of SMP8-PTHrP-R-overexpressing mice (line 127) showed a trend toward lower blood pressure compared with that in controls, although this difference was not significant (121 ± 3 vs. 127 ± 2 mm Hg; P > 0.05).



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  		Figure 2. Effect of overexpression of the SMP8-PTHrP-R transgene on mean blood pressure in awake mice. Blood pressure was measured by tail cuff in groups (n = 6) of transgenic and wild-type mice. The asterisk indicates a significantly lower blood pressure in transgenic animals compared with that in wild-type mice (117 ± 4 vs. 133 ± 3).

 
Cardiovascular hemodynamic measurements were also measured in anesthetized, closed chest mice (line 122), as shown in Fig. 3Go. Baseline blood pressure, mean velocity, and peripheral resistance were similar in both groups. The reason why baseline blood pressure was reduced in awake transgenic mice but not in anesthetized mice is unclear, but could relate to the decreased sympathetic tone induced by the anesthesia, which may have obscured any further reduction in pressure from local PTHrP. A similar result was observed in transgenic mice overexpressing PTHrP (18). The magnitude of the vasorelaxant response to increasing doses of PTHrP administered iv was significantly greater in the transgenic animals than in the controls. At the highest dose, blood pressure decreased to 36 ± 2 mm Hg in the transgenic mice and to only 45 ± 9 mm Hg in the controls (P < 0.05).



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  		Figure 3. Effect of overexpression of the SMP8-PTHrP-R transgene on cardiovascular hemodynamics in anesthetized mice. Mean arterial pressure (MAP), mean blood flow velocity, and total peripheral resistance (TPR) in wild-type (open circles) and SMP8-PTHrP-R transgenic (closed circles) mice before and during iv infusion with PTHrP-(1–34). Each point represents the mean of seven animals. Baseline MAP and TPR were not different between transgenic and wild-type mice. PTHrP-(1–34) NH2 produced dose-dependent decreases in MAP and TPR in both groups, but these changes were significantly greater in the PTH/PTHrP-R transgenic mice. The blood flow velocity did not differ between the two groups of animals, and thus, TPR responses mirrored those of MAP. Asterisks indicate a significant (P < 0.05) difference between transgenic and control mice.

 
Effects of overexpression of PTHrP-R on vascular contractility
The influence of overexpression of the SMP8-PTHrP-R on the contractile properties of mouse aorta was assessed in vitro in an organ bath. Endothelium-intact or denuded aortic rings from 9-week-old transgenic (line 122) and wild-type mice, isometrically mounted in the same bath, were precontracted with 1 µM phenylephrine, a dose that produced 80% maximal contraction. The maximal force of contraction and the concentration-response relationships elicited by phenylephrine were similar in transgenic and wild-type aortas (Fig. 4Go, A and C). PTHrP-(1–34) (10 nM) produced a more pronounced relaxation response in aortas from SMP8-PTHrP-R receptor mice than in those from wild-type mice. In wild-type mice, PTHrP-induced relaxation was greater in endothelium-intact aortas (Fig. 4Go, A and B), as we have reported previously (23). Figure 4BGo shows concentration-response relationships generated using a single concentration of PTHrP-(1–34) in endothelium-intact or denuded aortas from transgenic and wild-type mice. Interestingly, at the higher doses of PTHrP-(1–34), the relaxation response observed in transgenic mice was similar in endothelium-intact and denuded aortic preparations. Similar results were obtained in aortic ring preparations from a second line of SMP8-PTHrP-R-overexpressing mice (line 127), as summarized in Table 2Go.



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  		Figure 4. Effect of overexpression of the SMP8-PTHrP-R transgene on aortic contraction and relaxation responses. A, Representative force tracing from a PTHrP-induced relaxation of a phenlyephrine (PE)-precontracted aorta from a transgenic (TG) and a wild-type (WT) mouse. Endothelium-intact (+E) or denuded (-E) mouse aortas were isometrically mounted and contracted with 300 nM PE. Vessels were exposed to increasing concentrations of PTHrP-(1–34) NH2, and relaxation was monitored. B, PTHrP concentration-relaxation relationships of precontracted aortas with (closed symbols) or without (open symbols) endothelium from transgenic (square) and wild-type (circle) mice. Individual endothelium-intact mouse aortas were isometrically mounted and contracted with 300 nM PE. PTHrP was administered to generate relaxation relationships. Each data point represents the mean ± SEM of five separate aortas. C, Phenylephrine concentration-response curves for wild-type (circles) and transgenic (squares) mouse aortas. Endothelium-containing aortas were isometrically mounted, and cumulative phenylephrine concentration-response curves were generated. Each data point represents the mean ± SEM of three separate aortas. There was no significant difference between the concentration-response curves for transgenic and wild-type mice.

 

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  		Table 2. Contractile force and PTHrP-induced relaxant activity in aortas from PTH/PTHrP-R-overexpressing mice

 
Analysis of SMP8-PTHrP-R/PTHrP double transgenic mice
To examine the impact of overexpression of both PTHrP receptor and ligand in smooth muscle, F1 progeny from line 122 SMP8-PTHrP-R mice were bred with offspring from line 375 SMP8-PTHrP ligand-overexpressing mice as described previously (18). Genotyping of live offspring yielded no double transgenic mice, indicating that the overexpression of both PTHrP ligand and the PTH/PTHrP type 1 receptor in smooth muscle was embryonically lethal (Table 3Go). To determine the time of death during embryogenesis, timed pregnant females from matings of the receptor- and ligand-overexpressing transgenic mice were killed, and embryos were genotyped by PCR at various gestational time points. These studies demonstrated that embryos expressing both transgenes died at approximately embryonic day 9. Preliminary examination showed greatly enlarged hearts, with pericardial effusion and vascular pooling (Fig. 5Go). Ventricular contractions also appeared unsynchronized. Histological sections through double transgenic embryos revealed a reduced thickness of the ventricular wall, a lack of trabeculae, and prominent gaps between the cardiomyocytes.


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  		Table 3. Genotype frequency of offspring from crosses of SMP8-PTHrP- and SMP8-PTHrP-R-overexpressing mice

 


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  		Figure 5. A double transgenic SMP8-PTHrP/SMP8-PTHrP-R mouse embryo and a wild-type littermate are shown. A, Whole mounts on embryonic day 9.5 of double transgenic (left) and wild-type (right) embryos. The double transgenic embryo exhibits a greatly enlarged heart with pericardial effusion and vascular pooling (arrows). B, Histological sections of double transgenic (left) and wild-type (right) embryos on embryonic day 9.5. The trabeculae within the ventricular cavity (v) of the wild-type embryo are prominent (large arrows), whereas in the double transgenic embryo, trabeculae are severely reduced or absent (asterisks). Prominent gaps are also evident between the cardiomyocytes in the double transgenic hearts (small arrowheads). a, Atria. Bar, 100 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we describe the development of transgenic mice that selectively overexpress the PTH/PTHrP type 1 receptor in smooth muscle. Our primary objective for developing these mice and the PTHrP-overexpressing mice described in the accompanying report (18) was to investigate the role of PTHrP on cardiovascular function in its normal autocrine/paracrine setting in vivo. Previous in vitro studies have demonstrated that both PTHrP and the PTH/PTHrP type 1 receptor are expressed in vascular smooth muscle cells (24, 25) and in vessel preparations from several species (8). In addition, synthetic N-terminal PTHrP peptides that activate the PTH/PTHrP type 1 receptor in vascular smooth muscle cells and stimulate cAMP production (13) also relax vascular preparations (8). The development and characterization of these transgenic mouse models provide the first in vivo evidence that PTHrP functions as a local vascular smooth muscle vasodilator by activation of the PTH/PTHrP type 1 receptor.

Mice overexpressing the SMP8-PTHrP-R transgene in vascular smooth muscle demonstrated a significant reduction in systemic blood pressure. Therefore, overexpression of either PTHrP or the PTH/PTHrP type 1 receptor in vascular smooth muscle results in hypotension consistent with the postulated role of PTHrP as a local vasorelaxant. However, in these overexpression transgenic mouse models, the alterations in blood pressure probably represent an amplification of the physiological effects of PTHrP in the vasculature. Therefore, the hypotension seen in the receptor-overexpressing mice probably results from sustained activation of the overexpressed receptor by locally produced PTHrP. Theoretically, it is also possible that overexpression of the PTH/PTHrP-R leads to constitutive activation of adenylyl cyclase, which is known to result when other G protein-coupled receptors are overexpressed (26). However, such a scenario seems less likely in light of our previous studies that showed that massive overexpression of the PTH/PTHrP type 1 receptor in stably transfected vascular smooth muscle cells did not influence basal cAMP production (9).

Aortic vessel preparations from the SMP8-PTHrP-R mice exhibited a marked increase in sensitivity to the relaxant action of PTHrP-(1–34) NH2. Transgenic mice also had a greater reduction in blood pressure when infused with PTHrP-(1–34) NH2 than control mice. Therefore, the SMP8-PTHrP-R transgene appears to drive high level expression of functional receptors in vascular smooth muscle. In wild-type mice, the relaxant effects of PTHrP in aorta were much more pronounced when endothelium was present in the vessel wall. We have previously shown that the ability of PTHrP to relax mouse aorta is largely endothelium dependent, whereas an intact endothelium was not necessary for maximal relaxation of mouse portal veins (23). In these studies, the endothelium-dependent component of PTHrP-induced aortic relaxation was unaffected by pretreatment with either L-NNA or indomethacin, but was abolished by pretreatment with tetrabutyl ammonium, suggesting a requirement for an endothelium-derived hyperpolarizing factor. Interestingly, in the receptor-overexpressing mice, the pronounced relaxation responses induced by PTHrP were similar in both endothelium-denuded and endothelium-intact aortas, indicating that high level activation of this signaling pathway in smooth muscle masked the endothelium component of these responses.

The SMP8-PTHrP-R-overexpressing mice developed normally and had no gross abnormalities of any organ, aside from a modest increase in stomach weight. This is in contrast to the severe abnormalities in bone and heart observed in several of the transgenic mouse lines overexpressing SMP8-PTHrP described in the accompanying report (18). The variation in phenotype between ligand- and receptor-overexpressing mice may relate to the fact that transgenic PTHrP can diffuse and activate receptors expressed in adjacent cells. Thus, the hypercellular bone marrow and grossly increased bone volume in two SMP8-PTHrP transgenic mouse lines probably resulted from local production of transgenic PTHrP by stromal cells and activation of adjacent osteoblasts. By contrast, the targeted expression of the PTHrP-R remains restricted to smooth muscle and would be activated only when ligand is available.

The bone and heart abnormalities observed in several of the PTHrP-overexpressing transgenic mouse lines described in the companion report (18) as well as the relatively low numbers of founder mice obtained from multiple oocyte injections with the ligand expression construct suggested that high levels of PTHrP receptor activation at this gestational time point may have disrupted the development of these organs. To investigate this possibility, we created double transgenic mice that overexpressed both the ligand and receptor transgenes. All double transgenic mice died on approximately embryonic day 9.5 with evidence of major abnormalities in the developing heart. Although we have not formally established temporal and spatial expression of the SMP8 {alpha}-actin promoter in the mouse during embryogenesis, the native smooth muscle {alpha}-actin gene is known to be expressed in smooth muscle of the developing large blood vessels and in cardiac muscle on approximately embryonic day 10.5 in both mouse (27) and rat (28). Moreover, both PTHrP and the PTH/PTHrP type 1 receptor are expressed in rat fetuses on approximately day 11 (29), coincident with the time of heart and major blood vessel formation in the rat. We attribute the developmental abnormalities in the double transgenic mice to the effects of persistent activation of the PTH/PTHrP receptor at this gestational time point. It remains to be determined whether and to what extent this peptide plays a role in the normal development of the cardiovascular system. Mice with targeted disruption of the PTHrP gene survive until birth, suggesting that heart and blood vessel development can take place in the absence of the protein. On the other hand, Massfelder et al. (10) reported that the mitotic rate of aortic vascular smooth muscle cells in 18-day-old PTHrP null embryos was significantly reduced compared with that in PTHrP+/+ fetuses. These data together with the established importance of PTHrP and its receptor in the development of mammary gland (30), skin (31), and cartilage (32, 33) suggest that PTHrP also plays a role during the development of the cardiovascular system.

In summary, overexpression of the PTH/PTHrP type 1 receptor in vascular smooth muscle of transgenic mice reduces blood pressure in awake mice, probably through sustained activation of the receptor by endogenous ligand. To our knowledge, this study and the one described in the accompanying report are the first to demonstrate the outcome of expression and function of any paracrine vasoactive agent and its receptor in vascular smooth muscle of transgenic mice. Moreover, the similar cardiovascular phenotypes (i.e. hypotension) in both the ligand- and receptor-overexpressing mice strongly suggest that locally elaborated PTHrP acts through the classical PTH/PTHrP type I receptor in vascular smooth muscle to modulate vascular tone. Finally, the embryonic lethality and morphological features observed in mice overexpressing both PTHrP and its receptor suggest that PTHrP also participates in the morphogenesis of heart and perhaps the vasculature. Studies are now underway to formally characterize the defective cardiovascular abnormalities in these animals.


   Acknowledgments
 
The authors are grateful to Jianwei Wang and Hui Tang for technical assistance, and to Dr. Bill Scott for helpful discussion.


   Footnotes
 
1 This work was supported by NIH Grants HL-47811, HL-09781, T-32-HL-07571, and HL-43802. Back

Received August 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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