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Comparative Study
. 2010 Apr;298(4):H1146-54.
doi: 10.1152/ajpheart.00252.2009. Epub 2010 Feb 5.

Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases

Jianjie Wang  1 , Susan Bingaman, Virginia H Huxley
Affiliations
Comparative Study

Intrinsic sex-specific differences in microvascular endothelial cell phosphodiesterases

Jianjie Wang et al. Am J Physiol Heart Circ Physiol. 2010 Apr.

Abstract

The importance of gonadal hormones in the regulation of vascular function has been documented. An alternate and essential contribution of the sex chromosomes to sex differences in vascular function is poorly understood. We reported previously sex differences in microvessel permeability (P(s)) responses to adenosine that were mediated by the cAMP signaling pathway (Wang J, PhD thesis, 2005; Wang J and Huxley V, Proceedings of the VIII World Congress of Microcirculation, 2007; Wang J and Huxley VH, Am J Physiol Heart Circ Physiol 291: H3094-H3105, 2006). The two cyclic nucleotides, cAMP and cGMP, central to the regulation of vascular barrier integrity, are hydrolyzed by phosphodiesterases (PDE). We hypothesized that microvascular endothelial cells (EC) would retain intrinsic and inheritable sexually dimorphic genes with respect to the PDEs modulating EC barrier function. Primary cultured microvascular EC from skeletal muscles isolated from male and female rats, respectively, were used. SRY (a sex-determining region Y gene) mRNA expression was observed exclusively in male, not female, cells. The predominant isoform among PDE1-5, present in both XY and XX EC, was PDE4. Expression mRNA levels of PDE1A (male > female) and PDE3B (male < female) were sex dependent; PDE2A, PDE4D, and PDE5A were sex independent. Barrier function, P(s), was determined from measures of albumin flux across confluent primary cultured microvessel XY and XX EC monolayers. Consistent with intact in situ microvessels, basal monolayer P(s) did not differ between XY (1.7 +/- 0.2 x 10(-6) cm/s; n = 8) and XX (1.8 +/- 0.1 x 10(-6) cm/s; n = 10) EC. Cilostazol, a PDE3 inhibitor, reduced (11%, P < 0.05) P(s) in XX, not XY, cells. These findings demonstrate the presence and maintenance of intrinsic sex-related differences in gene expression and cellular phenotype by microvascular EC in a gonadal-hormone-free environment. Furthermore, intrinsic cell-sex likely contributes significantly to sexual dimorphism in cardiovascular function.

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Figures

Fig. 1.
Fig. 1.
A representative RT-PCR assay shows expression of SRY, a sex-determining region Y gene, in cultured endothelial cells (EC) derived from male (XY), but not in EC cells from female (XX), rats.
Fig. 2.
Fig. 2.
Expression of phosphodiesterase (PDE) mRNA in microvessel EC derived from rat skeletal muscles of males and females via real-time RT-PCR analysis (n = 8–11). A and B: fold changes in PDE expression normalized to PDE2A are given on the ordinate for cultured XY (A) or XX (B) endothelium, providing a comparison among the 5 PDE isoforms within EC of the same origin (XX or XY). If the value (A and B) is larger than 1, the expression level is greater than that for PDE2A mRNA, and vice versa. C and D: ΔΔCt values (C) defined as ΔCtXY cells subtracting ΔCtXX cells, and the fold differences (D) of PDE1–5 expression in XY cells normalized to XX EC. If the value is <0 in C or >1 in D, it indicates that this gene mRNA is expressed higher in XY EC relative to XX EC, and vice versa. P < 0.05 vs. #0 (C) and ★1 (A, B, and D). Numbers in bars (D) represent experimental replicates.
Fig. 3.
Fig. 3.
Comparable protein levels of PDE2A (A) and PDE4D (B) were expressed in EC from male (XY) and female (XX) EC. The representative immunoblot results for PDE2A and PDE4D are displayed. n represents the experimental numbers. β-actin serves as the controls for the amount of protein loaded for this assay.
Fig. 4.
Fig. 4.
The distribution of PDE2A and PDE4D in abdominal skeletal muscle microvessels was assessed by immunofluorescence staining and confocal laser scanning microscopy. PDE2A and PDE4D were detected by primary antibody, followed by secondary antibody conjugated with AlexaFluro 488 (green). Endothelium was identified with antibody against an EC marker, PECAM, and subsequently detected by secondary antibody conjugated with Alexa Fluro 568 (red). The yellow color is the superimposed image of the green and red images of PDE2A and PDE4D in microvascular endothelium. The bright-field images are from the tissue sections.
Fig. 5.
Fig. 5.
EC permeability to albumin in primary cultured male and female cells. Basal permeability Ps to albumin of EC derived from skeletal muscle microvessels of male and female rats did not differ with sex (A), while sexually dimorphic EC Ps responses to cilostazol, a PDE3 inhibitor, were observed (B). Relative EC Ps responses (normalized to its basal Ps) were expressed as a percentage of the changes (Ptest/Pcon − 1) × 100% (see materials and methods for explanation of equation terms). The numbers in the parentheses represent the experimental numbers. ★P < 0.05 compared with 0 (B).
Fig. 6.
Fig. 6.
Schematic of the mechanisms whereby PDEs mediate endothelial barrier integrity. PDE isoforms 2–5 are expressed constitutively in primary EC derived from rat skeletal muscle microvessels. PDE4 and -5 hydrolyze intracellular cAMP and cGMP, respectively. Both PDE2 and PDE3, hydrolyzing cAMP, are the critical molecules bridging between cGMP and cAMP. Their activity creates the cross talk, resulting in loosening and tightening the endothelial barrier via PKG and PKA pathways, respectively. PDE2 is activated, while PDE3 is inhibited by cGMP. Given that XX EC express higher levels of PDE3 and comparable levels of PDE2, -4, and -5 relative to XY EC, the intracellular levels of cAMP are expected to be elevated greatly in XX EC compared with XY EC. This appears to be the case only when the system is activated (e.g., when cilostazol is given to block PDE3-mediated cAMP degradation). Consequently, while basal Ps did not differ in monolayers of XX and XY EC, the decrease in Ps in response to cilostazol was greater in XX than XY EC. eNOS, endothelial nitric oxide synthase.
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