Analysis of short-term treatment with the phosphodiesterase type 5 inhibitor tadalafil on long bone development in young rats

Abstract

Cyclic GMP (cGMP) is an important intracellular regulator of endochondral bone growth and skeletal remodeling. Tadalafil, an inhibitor of the phosphodiesterase (PDE) type 5 (PDE5) that specifically hydrolyzes cGMP, is increasingly used to treat children with pulmonary arterial hypertension (PAH), but the effect of tadalafil on bone growth and strength has not been previously investigated. In this study, we first analyzed the expression of transcripts encoding PDEs in primary cultures of chondrocytes from newborn rat epiphyses. We detected robust expression of PDE5 as the major phosphodiesterase hydrolyzing cGMP. Time-course experiments showed that C-type natriuretic peptide increased intracellular levels of cGMP in primary chondrocytes with a peak at 2 min, and in the presence of tadalafil the peak level of intracellular cGMP was 37% greater ( P < 0.01) and the decline was significantly attenuated. Next, we treated 1-mo-old Sprague Dawley rats with vehicle or tadalafil for 3 wk. Although 10 mg·kg^-1·day^-1 tadalafil led to a significant 52% ( P < 0.01) increase in tissue levels of cGMP and a 9% reduction ( P < 0.01) in bodyweight gain, it did not alter long bone length, cortical or trabecular bone properties, and histological features. In conclusion, our results indicate that PDE5 is highly expressed in growth plate chondrocytes, and short-term tadalafil treatment of growing rats at doses comparable to those used in children with PAH has neither obvious beneficial effect on long bone growth nor any observable adverse effect on growth plate structure and trabecular and cortical bone structure.

Keywords: PDE5 inhibitor; bone; endochondral ossification; growth plate; young rat.

Fig. 1.

Tadalafil enhances the cGMP response to CNP in primary chondrocytes. A: qRT-PCR analysis of Pde isoforms in primary chondrocytes. n = 3 rats. B: time-courses of CNP-stimulated intracellular cGMP levels in primary chondrocytes with or without pretreatment of IBMX (250 µM) or tadalafil (50 µM). Cells were collected from indicated time points after CNP (100 nM) addition and analyzed by ELISA to measure intracellular cGMP amounts (n = 3). #P < 0.05, CNP + tadalafil vs. CNP; **P < 0.01, CNP + IBMX vs. CNP; ***P < 0.001, CNP + IBMX vs. CNP; ^aP < 0.05, CNP + IBMX vs. CNP + tadalafil by two-way ANOVA with Bonferroni’s post hoc test for multiple comparisons. cGMP, cyclic GMP; CNP, C-type natriuretic peptide; qRT-PCR, quantitative RT-PCR.

Fig. 2.

Tadalafil treatment increases the tissue cGMP level in young rats. A: rats were weighed weekly during the 3-wk tadalafil treatment period. n = 10 rats/group. B: ELISA analysis of cGMP amount in brain tissue of rats treated with vehicle, 2.5 mg/kg or 10 mg/kg tadalafil daily for 3 wk. n = 5 rats/group. C: ELISA analysis of cGMP amount in brain tissue of rats at 0, 3, 6, and 24 h after receiving 1 dose of 10 mg/kg tadalafil. n = 3 rats/time point. *P < 0.05, 2.5 mg/kg tadalafil vs. vehicle; **P < 0.01, 2.5 mg/kg tadalafil vs. vehicle; ^aP < 0.05, 10 mg/kg tadalafil vs. vehicle; ^bP < 0.01, 10 mg/kg tadalafil vs. vehicle; #P < 0.05 vs. 0 h by two-way (A) or one-way ANOVA (B and C) with Bonferroni’s post hoc test for multiple comparisons. cGMP, cyclic GMP; veh, vehicle.

Fig. 3.

Three weeks of tadalafil treatment does not affect longitudinal skeletal growth. A: tibia length of vehicle-treated and 10 mg·kg⁻¹·day⁻¹ tadalafil-treated rats was measured weekly by in vivo microcomputed tomography. n = 10 rats/group. B: length of tibial in rats after 3 wk of vehicle, 2 mg·kg⁻¹·day⁻¹, and 10 mg·kg⁻¹·day⁻¹ tadalafil treatments. n = 10 rats/group. C: representative H&E staining of growth plate from vehicle group and 10 mg·kg⁻¹·day⁻¹ tadalafil group. Double-arrowed lines point to the growth plate. D: thickness of the proliferative zone (Pro) and hypertrophic zone (Hyp) of the growth plate was quantified. n = 5 rats/group. E: H&E staining of chondral-ossification junction of vehicle-treated and 10 mg·kg⁻¹·day⁻¹ tadalafil-treated rats. Arrows point to erythrocytes within blood vessels that invade growth plate cartilage. F: H&E staining of tibial articular cartilage of vehicle-treated and 10 mg·kg⁻¹·day⁻¹ tadalafil-treated rats. Double arrowed lines point to the articular cartilage. Scale bar: 100 µm. Results were analyzed by two-way (A, D) or one-way (B) ANOVA with Bonferroni’s post hoc test for multiple comparisons. veh, vehicle.

Fig. 4.

Three weeks of tadalafil treatment does not change bone metabolism in the trabecular bone. A: representative 3-D reconstructed microcomputed tomography (microCT) images of tibial trabecular bone in rats treated with vehicle, 2.5 mg·kg⁻¹·day⁻¹ or 10 mg·kg⁻¹·day⁻¹ tadalafil. B: microCT measurement of trabecular bone structural parameters. n = 10 rats/group. C: osteoblast numbers (N.Ob) and osteoclast numbers (N.Oc) per bone surface (BS) were quantified by histomorphometric analysis. n = 5 rats/group. Results were analyzed by one-way ANOVA with Bonferroni’s post hoc test for multiple comparisons (B) or by unpaired t-test (C). 3-D, three-dimensional; BMD, bone mineral density; BV/TV, bone volume fraction; SMI, structure model index; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.

Fig. 5.

Cortical bone structure is not altered by 3 wk of tadalafil treatment. A: representative 3-D reconstructed microcomputed tomography (microCT) images of cortical bone in femoral midshaft region of rats treated with vehicle, 2.5 mg·kg⁻¹·day⁻¹ or 10 mg·kg⁻¹·day⁻¹ tadalafil. B: microCT measurement of cortical bone structural parameters. n = 10 rats/group. Results were analyzed by one-way ANOVA with Bonferroni’s post hoc test for multiple comparisons. Ct.Ar, corticol area; Ct.BMD, cortical tissue bone mineral density; Ct.Th; cortical thickness; Ec.Pm, endosteal perimeter; pMOI, polar moment of inertia; Ps.Pm, periosteal perimeter.