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Endocrinology Vol. 140, No. 4 1815-1825
Copyright © 1999 by The Endocrine Society

ARTICLES

Targeted Overexpression of Parathyroid Hormone-Related Protein (PTHrP) to Vascular Smooth Muscle in Transgenic Mice Lowers Blood Pressure and Alters Vascular Contractility1

Shigeto Maeda, Roy L. Sutliff, Jin Qian, John N. Lorenz, Jianwei Wang, Hui Tang, Toshiyuki Nakayama, Craig Weber, David Witte, Arthur R. Strauch, Richard J. Paul, James A. Fagin and Thomas L. Clemens

Division of Endocrinology, Departments of Medicine (J.Q., S.M., T.N., J.A.F., T.L.C.) and Molecular and Cellular Physiology (R.L.S., J.N.L., C.W., R.J.P., J.A.F., T.L.C.), University of Cincinnati (S.M., J.Q., R.L.S., J.N.L., J.W., H.T., T.N., C.W., R.J.P., J.A.F., T.L.C.), and the Department of Pathology (D.W.), Children’s Hospital, Cincinnati, Ohio 45267; and the Department of Cell Biology, Neurobiology, and Anatomy, Ohio State University (A.R.S.), Columbus, Ohio 43210

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) and its receptor are expressed in vascular smooth muscle cells and are believed to participate in the local regulation of vascular tone. To explore the function of locally produced PTHrP in vascular smooth muscle in vivo, we developed transgenic mice that overexpress PTHrP in smooth muscle using a smooth muscle {alpha}-actin promoter to direct expression of the transgene. In the PTHrP-overexpressing mice, messenger RNA expression was mainly restricted to smooth muscle-containing tissues. Several founders also expressed the transgene in bone and heart and exhibited striking abnormalities in the development of these tissues. In PTHrP-overexpressing mice, blood pressure was significantly lower than that in wild-type controls (121 ± 3 vs. 135 ± 2 mm Hg; P < 0.01). Moreover, the magnitude of the vasorelaxant response to iv infusions of PTHrP-(1–34)NH2 was significantly attenuated in the transgenic animals. A similar desensitization to PTHrP was observed in aortic ring and portal vein preparations. Surprisingly, PTHrP-overexpressing mice were also significantly less responsive to the hypotensive action of infused acetylcholine in vivo and to the relaxant actions of acetylcholine on aortic vessel preparations in vitro. In summary, we have successfully targeted overexpression of PTHrP to the smooth muscle of transgenic mice. When expressed in its normal autocrine/paracrine setting, PTHrP lowers systemic blood pressure and decreases vascular responsiveness to further relaxation by PTHrP and other endothelium-dependent vasorelaxants such as acetylcholine. We postulate that the heterologous desensitization to acetylcholine-induced relaxation in PTHrP-overexpressing blood vessels involves desensitization of second messenger/effector signaling pathways common to PTHrP and acetylcholine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH-RELATED protein (PTHrP) was originally identified as the tumor-derived peptide responsible for the syndrome of humoral hypercalcemia of malignancy (1). PTHrP and PTH share limited N-terminal sequence homology, which is sufficient to enable both proteins to activate a common G protein-linked receptor that is expressed in bone, kidney, and several other tissues (2). In contrast to PTH, which is produced exclusively in the parathyroid gland, PTHrP is expressed in a wide variety of normal fetal and adult tissues. The PTH/PTHrP receptor is frequently expressed in the same cells that produce PTHrP or in cells immediately adjacent to them (3). This spatial proximity of PTHrP and its receptor together with the fact that little if any PTHrP circulates under normal physiological conditions (4) suggest that PTHrP functions in an autocrine/paracrine fashion.

There is increasing evidence supporting a role for PTHrP in the control of vascular tone. PTHrP is expressed in blood vessels from a wide array of vascular beds, including rat aorta (5, 6), vena cava (5), kidney microvessels (7), arterial and venous supplies of the mammary gland (8), and serosal arterioles of the avian egg shell gland (9). The protein has also been detected in many fetal blood vessels (10, 11). In most if not all of these vessels PTHrP appears to be expressed in the smooth muscle layer. Exogenous application of synthetic PTHrP peptides exert relaxant activity on both conductance and resistance vessels from different species (12). Cultured arterial vascular smooth muscle cells express both PTHrP (13) and the type 1 PTH/PTHrP receptor (14). In addition, PTHrP production by cultured vascular smooth muscle cells is stimulated by vasoconstrictor agents such as angiotensin II (15), suggesting the existence of a short feedback loop through which the local vasorelaxant actions of PTHrP function to oppose pressor activity of angiotensin II and other vasoconstrictor agents. However, the biology of locally produced PTHrP in vascular or other smooth muscle beds has not previously been examined in vivo.

Transgenic models have recently been used to explore the autocrine/paracrine functions of locally produced PTHrP in the development of several organ systems in vivo. For example, transgenic mice with targeted overexpression of PTHrP in skin show marked disturbances in hair follicle development (16). In this same model, the targeting promoter (keratin 14) also directed PTHrP overexpression in myoepithelial cells of breast ductules, which resulted in failure of lactation in female transgenic mice (17). By contrast, the lack of PTHrP or its receptor in knockout mice demonstrated the importance of this peptide in chondrocyte differentiation (18, 19); both PTHrP and the PTH/PTHrP receptor knockouts are lethal and are associated with premature terminal differentiation and calcification of chondrocytes.

The object of the present studies was to determine the effects of locally produced PTHrP on vascular contractility by targeting its overexpression to smooth muscle of transgenic mice. Mice overexpressing PTHrP in vascular smooth muscle demonstrated a reduced systemic blood pressure compared with their wild-type littermates. Interestingly, the local overexpression of PTHrP also desensitized vascular beds to acetylcholine and sodium nitroprusside (SNP), suggesting heterologous desensitization of a common second messenger/effector pathway(s).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of transgenic mice
The SMP8-PTHrP chimeric gene was constructed by fusing a 3.6-kb fragment of the mouse smooth muscle {alpha}-actin gene (SMP8) containing 1074 bp of the 5'-flanking region, the 63-bp 5'-untranslated and 2.5-kb first intron, and 15 bp of exon 2 (20) to the human PTHrP complementary DNA (cDNA) (21) followed by the simian virus 40 early polyadenylation signal fragment (Fig. 1Go). The SMP8-PTHrP fusion gene was released from pSMP8-PTHrP by EcoRI and SphI restriction before microinjection. The male pronuclei of fertilized eggs from FVB/N mouse strains were microinjected with 2 pl linearized DNA 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 were 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 human PTHrP cDNA labeled by random priming (Prime-It II kit, Stratagene, La Jolla, CA). The transgene was identified as a unique 1.1-kb band. All animals received humane care in compliance with the local institutional animal care and use committee.



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  		Figure 1. Linear map of the SMP8-PTHrP fusion gene. The mouse SM {alpha}-actin promoter (top) consisted of -1074 bp of 5'-flanking region (light gray box), the transcription start site, 48 bp of exon 1 (black box), the 2.5-kb intron 1 (line), and 15 bp of exon 2 (black box) of the smooth muscle {alpha}-actin gene fused to a 0.9-kb rat PTHrP cDNA (dark gray box), followed by a 240-bp simian virus 40 early polyadenylation signal sequence (open box).

 
RNA isolation and Northern blot analysis
Total RNA was isolated from tissues by a single step acid guanidinium thiocyanate-phenol-chloroform extraction method. Northern blots were performed as previously described (13). Briefly, 20 µg tissue total RNA were gel separated, transferred to a Nylon membrane, and then hybridized with random primed human PTHrP cDNA. Loading was assessed by comparing the blots to the ethidium-stained ribosomal RNA.

Tissue histology and morphometry
Transgenic mice and their wild-type littermates were killed by CO2 asphyxiation. After determining the body weight, blood was collected by cardiac puncture, and serum was stored at -80 C until assay. Organs of interest were dissected, rinsed in ice-cold PBS, tissue-blotted, weighed, and immediately frozen in dry ice. Morphometry was performed using NIH Image version 1.61, an image-processing and analysis program for the Macintosh computer. The arterial section images of SMP8-PTHrP transgenic mice (line 375) and their age-matched wild-type controls were color-captured into the computer from Trichrome-stained sections through the microscope. After adjusting the image contrast, the area of interest was autooutlined, and the regions outside and inside the area were cleared as previously described (20). Cranial bone was fixed in 10% formalin-PBS (pH 7.4) at 4 C and decalcified in 10% formalin-PBS (pH 7.4) containing 20% EDTA. Paraffin blocks were prepared by standard histological procedures. Sections (5–6 µm thickness) were cut at several levels and stained with hematoxylin and eosin.

In situ hybridization
In situ hybridization was performed as previously described (22). Briefly, tissues dissected from animals at the indicated ages were fixed in 4% paraformaldehyde, saturated overnight with 30% sucrose in PBS, and frozen in M-1 (Lipshaw, Pittsburgh, PA). Cryostat sections (7 µm) were mounted on silane-coated slides. An antisense complementary RNA probe for human PTHrP was labeled with [35S]recombinant UTP using a commercially available kit (Stratagene, La Jolla, CA). For generation of the antisense PTHrP riboprobe, a PvuII to SacI fragment corresponding to nucleotides +230–537 of the human PTHrP cDNA (21) was cloned into the pBluescript SK(+) plasmid, linearized with EcoRI, and transcribed with T3 RNA polymerase. Hybridization was performed with a total of 1 x 106 cpm in a final volume of 30 µl/slide. The sections were hybridized overnight at 42 C, treated with 50 µg/ml ribonuclease A (Sigma Chemical Co., St. Louis, MO) and 100 U/ml ribonuclease T1 (Boehringer Mannheim, Indianapolis, IN) for 30 min at 37 C, and washed to a final stringency of 0.1 x standard citrate saline at 50 C. Slides were dipped in NTB2 emulsion (Eastman Kodak Co., Rochester, NY), diluted 1:1 with 0.6 mol/liter ammonium acetate, exposed for 10–14 days, and developed in D19 developer (Eastman Kodak Co.). Sections were photographed under darkfield illumination.

Noninvasive blood pressure measurements
After a 5-day training period, daily blood pressure measurements were performed in conscious mice over a 5-day period using a computerized tail-cuff system (Visitech Systems, Apex, NC). Animals were placed in a Lucite restrainer with their tails protruding from a small opening in the back. A balloon cuff was placed over the proximal portion of the tail, and a more distal portion of the tail was draped over a photoelectric sensor for detecting blood flow. For 10 consecutive cycles, the balloon cuff was inflated by a small air pump until detectable blood flow in the tail ceased; this pressure was taken as the end point. The cuff was then immediately deflated, and the next cycle was started 10 sec later. In each trial, the 10 measurement cycles were preceded by 10 preliminary cycles to acclimatize the mice to the apparatus.

Measurement of mean arterial pressure and cardiac output in intact mice
The surgical preparation for determining circulatory parameters in the intact mouse was adapted from that described previously in detail (23). In brief, mice were anesthetized with an ip injection of ketamine (50 µg/g BW) and inactin (100 µg/g BW; Research Biochemicals International, Natick, MA). The right femoral artery and vein were cannulated with hand-drawn polyethylene tubing (o.d., ~0.3 mm) for the measurement of arterial pressure and the infusion of experimental agents. The arterial catheter was connected to a low compliance transducer (COBE CDXIII, Arvada, CO), and amplified pressure signals were recorded using a MacLab 4/s data acquisition system at a sampling rate of 1000 samples/sec. A 0.5-mm catheter custom fashioned with a 20-MHz Doppler crystal at the tip was then inserted into the right carotid artery and advanced to its junction with the ascending aorta. The catheter was connected to a Millar MDV-20 pulsed Doppler velocimeter (Houston, TX), and signals for phasic and mean flow were recorded as an estimate of cardiac output. The position of the catheter was constantly adjusted so as to obtain a maximum value for peak aortic blood flow velocity. We have previously demonstrated that the phasic and mean signals from the Doppler crystals closely reflect those obtained simultaneously from a bulk flow meter positioned around the ascending aorta in the opened chest mouse (unpublished observation). In addition, the values obtained for peak and mean flow velocity using the pulsed Doppler approach are consistent with other measurements of cardiac output in the mouse (24, 25). Beat to beat values for mean arterial pressure and mean aortic blood flow velocity were used to calculate a tracing of total peripheral resistance with the units millimeters per Hg/cm·sec. 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.

Dose-response relationships for PTHrP and acetylcholine were determined in each group of animals by infusing increasing concentrations of these peptides, dissolved in saline, at a constant rate of 0.1 µl/min·g BW for 3 min. Average reported values for each variable were calculated from a 20-sec period occurring at the maximum response of TPR for each dose. The animal was allowed to recover fully for a 10- to 15-min period before administration of each subsequent dose.

Analysis of blood vessel contractile properties
Blood vessel contractile properties were analyzed as described previously (26, 27) with modifications. Briefly, 9-week-old FVB/N mice and age-matched SMP8-PTHrP transgenic mice were killed in a charged CO2 chamber. Segments of thoracic aorta (5–7 mm) were dissected and mounted for isometric force recording as previously described (26). The portal vein was dissected from the mouse by tying a 4–0 suture at the two ends of the vessel (between the hepatic bifurcation and the anterior mesenteric vein). The portal vein was cut free, and each end was secured with its thread to the myograph. From the time of dissection the vessel was maintained in a physiological salt solution containing 118 mmol/liter NaCl, 4.73 mmol/liter KCl, 1.2 mmol/liter MgCl2, 0.026 mmol/liter EDTA, 1.2 mmol/liter KH2PO4, 2.5 mmol/liter CaCl2, and 5.5 mmol/liter glucose buffered with 25 mmol/liter NaH2CO3 that when bubbled with 95% O2-5% CO2 was pH 7.2 at 37 C. Experiments were conducted at optimal tension by adjusting the length of the vessels to a point where maximum peak to peak oscillations were observed. Force measurements were obtained using a Harvard Apparatus differential capacitor force transducer (South Natick, MA), which was connected to a Biopac MP100 data acquisition system. Data from concentration-response curves were compared using two-way ANOVA. Significance was defined as P < 0.05 for all tests.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of SMP8-PTHrP mice and examination of transgene expression
Four SMP8-PTHrP founder animals were obtained from a total of over 155 screened. This is a very low number compared with that obtained after implantation of eggs microinjected with SMP8-insulin-like growth factor I (SMP8-IGF-I) (22) or PTHrP receptor as described in the accompanying report (28). One SMP8-PTHrP founder mouse developed tachypnea with failure to thrive and died 3 weeks after birth. Histological analysis of necropsy material showed calcification of the atrium of the heart and lung capillary hemorrhage. Two additional founders (lines 368 and 383) exhibited a knobby tail and an increase in bulk of the hind limbs that severely restricted their mobility. Attempts to mate these founders yielded only two positive F1 offspring that were ultimately killed. There were no obvious histological defects in the reproductive organs of these mice, suggesting that their reproductive defect was primarily due to reduced mobility and discomfort caused by the bone abnormality. Histological analysis of these mice from both lines revealed a similar bone phenotype that was characterized by grossly increased bone volume and fibrous dysplasia of marrow cavities in both membranous and endochondral bone (Fig. 2Go). The difficulty in obtaining viable mice overexpressing PTHrP in smooth muscle together with the variable phenotypes of the founders obtained indicate that high level expression of PTHrP in critical tissues disrupts development and in some cases causes embryonic death. F1 offspring from an additional line (375) exhibited no obvious outward phenotype and expressed high levels of PTHrP messenger RNA (mRNA) in smooth muscle. These mice were therefore propagated for study of the effects of PTHrP overexpression on their cardiovascular function.



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  		Figure 2. Histological sections of decalcified hematoxylin/eosin-stained cranial bones from a wild-type and an SMP8-PTHrP 368 transgenic mouse. A and B show low magnification calvarial sections (x2) from the wild-type and the transgenic mouse, respectively, illustrating the massively increased cranial bone volume in the transgenic mouse (B). C shows a higher magnification (x40) of a cranial section from the transgenic mouse, demonstrating a striking increase in marrow fibrosity and evidence of increased numbers of osteoblasts (small arrows) and osteoclasts (large arrows).

 
Abundance and localization of PTHrP mRNA expression
The tissue distribution of endogenously produced and transgenic PTHrP mRNA in F1 progeny from line 375 is shown in Fig. 3AGo. Endogenous PTHrP mRNA was present at low levels in bladder and stomach, but was undetectable in other tissues. The PTHrP transgene, identified because of its slightly smaller size (1.1 kb) compared with the endogenous PTHrP mRNA (1.4 kb), was detected at very high levels in bladder, stomach, uterus, and aorta and to a lesser degree in intestine. Figure 3BGo shows a Northern blot subjected to phosphorimaging for an extended period to assess the relative expression of transgenic PTHrP mRNA in different tissues of two individual SMP8-PTHrP transgenic mice. In the mouse from line 375, transgenic mRNA was expressed at high levels in smooth muscle and at lower levels in heart. The mouse from line 368 that exhibited the bone phenotype described above expressed comparatively higher levels of the transgene in bone and heart.



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  		Figure 3. A, Northern blot analysis of SMP8-PTHrP transgene expression in tissues from a representative transgenic mouse (line 375) and a wild-type littermate. Twenty micrograms of total RNA were gel separated, transferred to a nylon membrane, and then hybridized with a human PTHrP cDNA (top panel). The far right lane shows RNA from a human squamous carcinoma cell line (HTB35), which was used as a marker for the endogenous 1.4-kb mRNA species (arrow). The PTHrP transgene mRNA, which was distinguished from the endogenous transcript by its slightly smaller size (1.1 kb), was expressed at high levels in SMC-rich tissues. There was also detectable transgene mRNA expression in heart. The lower panel shows the ethidium bromide staining to indicate the equivalence of RNA loading. B, SMP8-PTHrP mRNA transgene expression in tissues from two separate SMP8-PTHrP transgenic lines (368 and 375). Transgene mRNA was predominately expressed in smooth muscle-rich tissues, although there was detectable expression in bone and heart in both lines. Line 368, which exhibited the skeletal phenotype, expressed higher levels of PTHrP mRNA in bone. The bottom panel shows the ethidium bromide-stained gel. The level of transgene expression normalized to the ethidium signal is given below each lane.

 
Transgene mRNA was localized in smooth muscle-rich tissues of line 375 by in situ hybridization with a human-specific riboprobe (Fig. 4Go). Transgene mRNA expression was largely confined to smooth muscle, including the smooth muscle cell layers of the intestine, aorta, bladder, stomach, bronchus, pulmonary artery, uterus, and meningeal and renal arterioles. The PTHrP transgene was also expressed in the stromal cells of the lamina propria of the gut, which are known to express endogenous smooth muscle {alpha}-actin (29).



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  		Figure 4. Localization of the PTHrP transgene mRNA in SMP8-PTHrP transgenic mice by in situ hybridization. A, Section of small intestine from a transgenic mouse line hybridized with the antisense human PTHrP riboprobe. There is a strong signal in the smooth muscle cells of the muscularis propria (MP) of the bowel and in the stromal cells of the lamina propria (arrows). B, Section of small intestine from a wild-type mouse that was also hybridized with the human PTHrP probe. No signal is detected in the wild-type animals. The yellow-green color in the crypts of the mucosal glands in both the transgenic and wild-type mouse tissue is autofluorescence from the Paneth cell granules and does not represent hybridization signal. C, Tangential section through the aorta of a transgenic mouse. Strong signals were also evident in brain meningeal arterioles (D) and in smooth muscle layers of the uterus (E) and bladder (F). G shows that the human PTHrP riboprobe signal was also localized to the smooth muscle cell layers in the wall of the bronchus (br) and pulmonary artery (pa) of the lung. H shows a section through the kidney, with a strong signal evident in an arteriole. Images are darkfield. Magnification: A–G, x100; H, x400.

 
Total body and organ weights of SMP8-PTHrP transgenic mice
Total body, carcass, and individual organ weights were determined in F1 mice derived from line 375 at 9 and 13 weeks of age (Table 1Go). For unexplained reasons, total body and carcass weights of female transgenic mice were significantly lower than those of their wild-type littermates. These differences tended to diminish with advancing age. Even after adjusting for body weight, intestinal weight was decreased at both 9 and 13 weeks of age, whereas stomach weight was greater in 13-week-old male and female transgenic mice. There were no major differences in the weights of any other organs (Table 1Go).


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  		Table 1. Total body and allometry of SMP8-PTHrP mice and age-matched nontransgenic controls

 
Histomorphometry of transverse sections taken at identical sites from aorta showed no significant differences in outer perimeter, luminal area, or medial area or thickness in transgenic vs. wild-type mice (data not shown). Serum total calcium measured in 9-week-old SMP8-PTHrP male mice was not significantly different in the PTHrP-overexpressing mice compared with aged-matched control mice (10.5 ± 0.2 vs. 10.4 ± 0.12 mg/dl). This indicates that PTHrP overexpression remained confined within the local smooth muscle compartment, as increases in systemic PTHrP would be expected to cause hypercalcemia (30).

Effects of overexpression of PTHrP on blood pressure and hemodynamics in the whole animal
We next investigated whether overexpression of PTHrP influenced cardiovascular hemodynamics in the intact mouse. Blood pressure was evaluated for 5 days by tail cuff sphygmomanometry in four wild-type and six transgenic littermates. Blood pressure was significantly lower in the transgenic animals compared with that in wild-type mice (121 ± 3 vs. 135 ± 2 mm Hg; P < 0.01). Cardiovascular hemodynamic measurements were also made in anesthetized, closed chest mice, as shown in Fig. 5Go. In contrast to the results obtained in conscious mice, baseline blood pressure did not differ significantly between the two groups. Baseline mean velocity and peripheral resistance were also similar in the two groups. The lack of a difference in baseline blood pressure and total peripheral resistance in transgenic and wild-type anesthetized mice may relate in part to the decreased sympathetic tone induced by the anesthesia, which could have obscured any further reduction in pressure by local overexpression of PTHrP.



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  		Figure 5. Effect of PTHrP overexpression on cardiovascular hemodynamics in anesthetized mice. Arterial pressure (top panel), mean blood flow velocity (middle panel), and total peripheral resistance (bottom panel) in wild-type and SMP8-PTHrP transgenic mice (n = 7) before and during iv infusion with PTHrP-(1–34)NH2 are shown. Baseline blood pressures (shown in the open circles) drifted upward in both groups of animals throughout the dose-response protocol. Nonetheless, the change in blood pressure in response to infusion with PTHrP was significantly attenuated in transgenic animals compared with that in wild-type mice. Blood flow velocity did not differ between the two groups of animals; thus, total peripheral resistance responses mirrored those of mean arterial pressure. *, P < 0.05 compared with baseline; {dagger}, P < 0.05 compared with the wild-type response.

 
Infusion of increasing doses of PTHrP-(1–34)NH2 into transgenic and wild-type mice resulted in an increase in heart rate and a reduction in blood pressure in both groups. Heart rate increased by a similar degree in wild-type (382 ± 16 to 480 ± 18 beats/min) and transgenic (383 ± 20 to 497 ± 31 beats/min) mice consistent with the previously documented positive chronotropic effect of PTHrP in several species (12). However, the magnitude of the vasorelaxant effect in response to increasing doses of iv infused PTHrP-(1–34)NH2 was blunted in the transgenic animals. At the highest dose, blood pressure decreased to 46.4 ± 2.5 mm Hg in the wild-type animals and to only 69.7 ± 7.6 mm Hg in the transgenic mice. Baseline blood pressure drifted upward in both groups of animals throughout the dose-response protocol. Nonetheless, the change in blood pressure in response to infusion of PTHrP-(1–34)NH2 was consistently attenuated in the transgenic animals; the maximum change was 36.6 ± 1.0 in the wild-type mice and 23.0 ± 0.8 in the transgenic animals (P < 0.05). As the mean velocity did not differ between the two groups of animals, the total peripheral resistance responses mirrored those of mean arterial pressure.

Because the ability of PTHrP to maximally relax murine aorta requires the presence of an intact endothelium (31), we investigated the possibility that local overexpression of PTHrP would also alter relaxation responses to other vasodilators that act through endothelium-dependent mechanisms. Acetylcholine was infused over a 30-min period at stepwise dose increments ranging from 0.3 ng/ml to 0.3 mg/ml. The maximal change in pressure in response to acetylcholine infusions was also blunted in animals overexpressing PTHrP. At the highest dose, acetylcholine decreased blood pressure by 51.7 ± 4.8 mm Hg in the wild-type animals and by only 33.1 ± 5.3 mm Hg in the transgenic mice. Again, peripheral resistance mirrored pressure changes; mean changes were 5.3 ± 0.8 mm Hg/cm·sec in the wild-type and 3.8 ± 0.2 mm Hg/cm·sec in the transgenic mice.

Effects of overexpression of PTHrP on vascular contractility
To further investigate the effect of overexpression of PTHrP on vascular tone, we analyzed the contractile properties of PTHrP in aorta and portal vein preparations from 9-week-old mice. Aortic rings from transgenic and wild-type mice, isometrically mounted in the same bath, were precontracted with 1 µM phenlyephrine, a concentration that produced an 80% maximal contraction. There was no significant difference in the maximal force of contraction elicited by phenlyephrine in transgenic vs. wild-type aortas (Fig. 6AGo). A similar result was obtained when the vessels were precontracted by depolarization with KCl (not shown). In aortas from wild-type mice, PTHrP-(1–34)NH2 produced marked relaxation responses, whereas there was no effect on aortas from PTHrP-overexpressing mice (Fig. 6AGo). Relaxation-response curves were generated using a single concentration of PTHrP per aorta, because addition of a single dose of PTHrP-(1–34)NH2 was associated with desensitization to subsequent doses of PTHrP (Fig. 6BGo). Consistent with the in vivo results, PTHrP-(1–34)NH2 produced a dose-dependent relaxation of wild-type mouse aortas, but had virtually no relaxant effect on the aortas from the PTHrP-overexpressing mice, even at concentrations as high as 100 nM. This finding suggests that the local overexpression of PTHrP resulted in desensitization of the aorta to PTHrP. A similar phenomenon was observed in the portal vein. PTHrP induced dose-dependent relaxation of spontaneous contractions in both transgenic and wild-type mouse portal vein (Fig. 7Go). Comparison of the concentration-response relationships revealed that portal veins from PTHrP-overexpressing mice were more resistant to PTHrP-induced relaxation than those from wild-type counterparts.



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  		Figure 6. A, PTHrP-induced relaxation of the aorta from transgenic (TG) and wild-type (WT) mice. Endothelium-intact mouse aortas were isometrically mounted and contracted with 300 nM phenylephrine (PE). A maximal dose of PTHrP-(1–34)NH2 (30 nM) was added, and relaxation was monitored. B, PTHrP concentration-relaxation relationships of precontracted aortas from transgenic ({blacksquare}) and wild-type (•) 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 percent relaxation (mean ± SEM) observed in six individual aortas.

 


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  		Figure 7. Desensitization of portal vein to PTHrP-evoked relaxation in mice overexpressing PTHrP. Relaxation response tracings are shown in representative portal veins from wild-type (top panel) and transgenic (bottom panel) mice. Vessels were isometrically mounted, as described in Materials and Methods, and exposed to increasing concentrations of PTHrP-(1–34)NH2. The tracing is representative of three comparable experiments.

 
As overexpression of PTHrP was associated with a reduced responsiveness to the relaxant activity of acetylcholine in vivo, we next compared the effect of this vasodilator on activity of aortas from wild-type and PTHrP-overexpressing mice. In agreement with the in vivo results described above, aortas from PTHrP-overexpressing mice precontracted with phenlyephrine exhibited a significantly reduced relaxation response to acetylcholine compared with those observed in the wild-type controls (Fig. 8Go). The EC50 values were 7.9 ± 1.1 and 33 ± 2 nM for wild-type and transgenic mice, respectively. Interestingly, SNP, which exerts its vasorelaxant properties through the generation of nitric oxide (NO), also elicited significantly less relaxant activity in the PTHrP-overexpressing aortas (Fig. 8BGo). EC50 values were 6.5 ± 1.1 and 31 ± 5 nM for wild-type and transgenic mice, respectively.



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  		Figure 8. Aortas from SMP8-PTHrP-overexpressing mice are desensitized to acetylcholine- and SNP-induced relaxation. Endothelium-intact aortic rings from transgenic ({blacksquare}) and wild-type (•) mice were isometrically mounted and contracted with 1 µM phenylephrine. Concentration-relaxation relationships are shown for acetylcholine (A) and SNP (B). Each data point represents the mean ± SEM of the relaxation force observed in five individual aortas.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of PTH to lower blood pressure has been known for decades, yet its physiological significance as a regulator of vascular tone has been the subject of much debate (12). The discovery of PTHrP and the demonstration that it could replicate vasorelaxant actions of PTH by activation of a common receptor suggested that PTHrP, rather than PTH, functioned in a local autocrine mode to regulate vascular smooth muscle cell tone. In these studies we characterized the effects of locally expressed PTHrP on cardiovascular hemodynamics and vascular reactivity using transgenic mice that overexpressed PTHrP in vascular smooth muscle.

The SMP8 promoter was selected to target PTHrP to smooth muscle because its natural gene product, smooth muscle {alpha}-actin, is expressed exclusively in smooth muscle of the adult mouse. The -1074 bp flanking region of the mouse smooth muscle {alpha}-actin promoter contains a conserved sequence that represses expression in nonmyogenic fibroblast cells, and a serum-response element and six E box motifs that confer high level expression in aortic smooth muscle cells (32). The identical segment of this promoter has recently been used to drive high level expression of IGF-I (22) and IGF-binding protein-4 (20) to the smooth muscle of transgenic mice. In these studies, the expression of both IGF-I and IGF-binding protein-4 transgene constructs was entirely confined to smooth muscle-rich tissues, precisely mimicking the pattern of expression of the endogenous smooth muscle {alpha}-actin gene. By contrast, the SMP8-PTHrP transgene construct was expressed to variable degrees in nonsmooth muscle tissues, particularly in adult heart and bone, which do not express smooth muscle {alpha}-actin. However, smooth muscle {alpha}-actin is transiently expressed in skeletal and cardiac muscle during embryogenesis (33, 34). It is possible that increased PTHrP in these tissues during embryogenesis may have delayed the developmental program of cardiomyocytes and bone marrow stromal cells, leading to persistence of an embryonic pattern of smooth muscle {alpha}-actin in adult tissues.

It is noteworthy that the skeletal phenotype observed in two separate SMP8-PTHrP lines is similar to that previously described in transgenic mice overexpressing PTHrP in growth plate chondrocytes (35). The Col II-PTHrP mice demonstrated a hypercellular fibrous marrow immediately adjacent to primary spongiosa reminiscent of osteitis fibrosa. As smooth muscle {alpha}-actin is known to be expressed in a subpopulation of bone marrow stromal cells (36), its local production would be expected to greatly accelerate bone turnover and give rise to a bone phenotype resembling osteitis fibrosa. Unfortunately, these mice did not reproduce, and they could not be characterized in more detail. Nonetheless, the histopathological abnormalities evident in heart and bone of these lines of SMP8-PTHrP transgenic mice are probably directly attributable to the local effects of PTHrP. In addition, the relatively low number of viable PTHrP-overexpressing mice obtained suggests that massive overexpression during embryogenesis may have important deleterious effects on development (28).

Transgenic mice from line 375 developed with no apparent physical abnormality and expressed the SMP8-PTHrP transgene predominantly in smooth muscle, as revealed by in situ hybridization. Measurements of cardiovascular function and characterization of vascular contractile properties in these mice provide the first in vivo evidence for a local vasorelaxant role for PTHrP. PTHrP-overexpressing mice demonstrated a significantly reduced systemic blood pressure compared with wild-type mice. This is remarkable considering the multiple compensatory mechanisms that would be expected to be triggered to maintain cardiovascular homeostasis. We recognize, however, that alterations in blood pressure in the transgenic mice occurred as the result of enforced overexpression of PTHrP and, by definition, probably represent an amplification of the physiological effects of PTHrP in the vasculature. However, the conclusion that the reduction in blood pressure is due to the overexpression of PTHrP is further strengthened by the fact that targeted overexpression of the PTH/PTHrP receptor to the smooth muscle of transgenic mice also results in animals with lowered blood pressure (28). Finally, as low levels of SMP8-PTHrP were also expressed in the brain, we cannot rule out the possibility that a component of the hypotension occurred as the result of a central effect of PTHrP.

PTHrP-overexpressing mice displayed decreased sensitivity to the effects of exogenous PTHrP on blood pressure and total peripheral resistance in vivo as well on vasorelaxation responses in organ bath preparations of aorta and portal vein. This finding is compatible with desensitization of PTH/PTHrP receptor/effector coupling mechanisms by high level expression of PTHrP within the vessel wall, as has been previously demonstrated in cultured bone and vascular smooth muscle cells (14) and murine aorta preparations (31). Although we did not attempt to directly measure receptor abundance in the transgenic mouse vasculature, Northern blot analysis of aorta, bladder, and other smooth muscle-rich tissues from these mice showed no significant changes in PTH/PTHrP receptor mRNA abundance (not shown). In addition, PTHrP-overexpressing aortas were less responsive to acetylcholine and SNP, which indicates that the mechanisms involved in the desensitization process include signaling pathways distal to the PTH/PTHrP receptor. Acetylcholine relaxes mouse aorta through endothelium-dependent NO formation, whereas SNP directly stimulates NO production in vascular smooth muscle. The mechanisms responsible for PTHrP-induced vasorelaxation remain incompletely characterized, but in the aorta they clearly depend on the presence of an intact endothelium (31). Therefore, the fact that overexpression of PTHrP desensitizes mouse vessels to the effects of both acetylcholine and SNP indicates that the signal transduction cascades of PTHrP and NO-mediated vasorelaxants converge on a common distal effector pathway.

In summary, we have selectively expressed PTHrP in smooth muscle of transgenic mice, representing the first example of successful targeting of a vasoactive agent in this tissue. When overexpressed in its normal paracrine setting, PTHrP reduces systemic blood pressure and desensitizes the vessels to further effects of exogenous PTHrP, acetylcholine, and possibly other vasorelaxants that act via a NO pathway. This transgenic mouse model should prove useful to further explore the functions of locally produced PTHrP in the cardiovascular system as well as in other smooth muscle-rich tissues such as bladder, stomach, and intestine.


   Acknowledgments
 
The authors thank Lisa Artmayer, Pam Groen, and Kathy Saalfeld for technical assistance, and Alicia Emily for photographic assistance. The human PTHrP cDNA was generously provided by Dr. Larry Suva.


   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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