Microvascular Sex- and Age- Dependent Phosphodiesterase Expression

Abstract

Objective: The cyclic nucleotide second messengers, cAMP and cGMP, are pivotal regulators of vascular functions; their cellular levels are tightly controlled by the cyclic nucleotide hydrolases, phosphodiesterases (PDE). Biologic sex and age are recognized as independent factors impacting the mechanisms mediating both vascular health and dysfunction. This study focused on microvessels isolated from male and female rats before (juvenile) and after (adult) sexual maturity under resting conditions. We tested the hypothesis that sexual dimorphism in microvascular PDE expression would be absent in juvenile rats, but would manifest in adult rats. Methods: Abdominal skeletal muscle arterioles and venules were isolated from age-matched juvenile and adult male and female rats under resting conditions. Transcripts of five PDE families (1-5) associated with coronary and vascular function with a total of ten genes were measured using TaqMan real-time RT-PCR and protein expression of microvessel PDE4 was assessed using immunoblotting and immunofluorescence. Results: Overall expression levels of PDE5A were highest while PDE3 levels were lowest among the five PDE families (p < 0.05) regardless of age or sex. Contrary to our hypothesis, in juveniles, sexual dimorphism in PDE expression was observed in three genes: arterioles (PDE1A, female > male) and venules (PDE1B and 3A, male > female). In adults, gene expression levels in males were higher than females for five genes in arterioles (PDE1C, 3A, 3B, 4B, 5A) and three genes (PDE3A, 3B, and 5A) in venules. Furthermore, age-related differences were observed in PDE1-5 (in males, adult > juvenile for most genes in arterioles; in females, adult > juvenile for arteriolar PDE3A; juvenile gene expression > adult for two genes in arterioles and three genes in venules). Immunoblotting and immunofluorescence analysis revealed protein expression of microvessel PDE4. Conclusion: This study revealed sexual dimorphism in both juvenile and adult rats, which is inconsistent with our hypothesis. The sex- and age-dependent differences in PDE expression implicate different modulations of cAMP and cGMP pathways for microvessels in health. The implication of these sex- and age-dependent differences, as well as the duration and microdomain of PDE1-5 activities in skeletal muscle microvessels, in both health and disease, require further investigation.

Keywords: adult; age; arterioles; juvenile; phosphodiesterase; sex; skeletal muscle; venules.

FIGURE 1

Expression level of PDE1-5 transcripts in adult and juvenile skeletal muscle arterioles and venules of male and female rats. Arterioles and venules were isolated from abdominal skeletal muscles of four group rats: adult males (AM), adult females (AF), juvenile males (JM), and juvenile females (JF). Microvessel PDE 1-5 mRNA expression was measured by using TaqMan real-time RT-PCR assay. (A) The fold-change relative to β-actin for PDE gene expression was calculated as 2^−ΔCt, where ΔCt = Ct_{target gene}–Ct_β-actin. PDE5A mRNA levels were the highest among ten genes in arterioles and venules in each group (n = 4–7, ^# p < 0.05). The order of expression levels in PDE1 family: 1A≈1C > 1B; likely for PDE4 family, 4B ≈ 4D > 4A. Expression levels of PDE3 family were relatively low vs. other families, in which there was no significant difference between PDE3A and PDE3B although there was a trend of low expression for PDE3A. These expression patterns were similar for both arterioles and venules in four group rats. (B) Sex-specific difference in PDE 1-5 mRNA levels. The relative fold-change between males and females were calculated by 2^−ΔΔCt, where ΔΔCt = ΔCt_{(high expression)}–ΔCt_{(low expression)}. In arterioles, levels of PDE1C, 3A, 3B, 4B, 5A were greater in adult males compared with adult females (i); PDE1A level was greater in juvenile females compared with juvenile males (ii). In venules, expression levels of PDE3A, 3B, and 5A were higher in adult males compared with adult females (iii); PDE1B and 3A were higher in juvenile males compared with juvenile females (iv). (C) Reproductive maturity-specific difference in PDE 1-5 mRNA level. Using the similar analysis to sex-specific difference, the fold-change between adult and juvenile with the same sex was expressed as 2^−ΔΔCt. In arterioles, expression levels of PDE1C, 3A, 3B, 4A were greater for adult males compared with juvenile males (i); PDE1A and 1C expression for juvenile females exceeded the counterparts for adult females and only PDE3A level was greater for adult females relative to juvenile females (ii). In venules, PDE4D was greater in adult males relative to juvenile males (iii) whereas adult females expressed higher levels of PDE1B, 4A, and 4D relative to juvenile females (iv). The data are means ± SEM of fold-changes. Experimental replication was 4–7 (n = 4–7) and * indicates p < 0.05.

FIGURE 2

PDE4 protein expression in skeletal muscles arterioles and venules. (A) The representative immunoblotting using non-selective PDE4 antibody shows PDE4 protein expressed in arterioles and venules isolated from rat abdominal skeletal muscles. Rat brain was used as a positive control. The expression of β-actin protein was used as a loading control for corresponding vessels. Art: arteriole; Ven: venule. (B) The representative immunofluorescence imaging was acquired using scanning confocal microscopy. The sectioned rat abdominal skeletal muscle was stained with PDE4A primary and Alexa-488 (green color) secondary antibodies (i) as well as CD31 (an endothelial marker) primary and Alexa-568 (red color) secondary antibody (ii). The images of (i) and (ii) were superimposed (iii), in which the yellow color indicates co-localization of PDE4A and CD31. The section in the small box of image c was enlarged (iv), showing the co-localization of PDE4A and CD31 molecules. The transmitted light image (v) shows the venule of skeletal muscle tissue. The scale bar represents 50 µm for all the images except the image (iv).

FIGURE 3

Heat-map comparison for expression levels of PDE1-5 in both arterioles and venules across adult and juvenile male and female rats. For each gene, expression level was compared across all microvessels regardless of its origin (age, sex, and microvessel type) by normalizing each expression level to the highest mRNA level (2^−∆∆Ct, where ∆∆Ct = ∆Ct_each–∆Ct_highest). The color is defined as the highest expression level in green (100%) and the lowest in yellow (0%). M: male; F: female.