Greater fractalkine expression in mesenteric arteries of female spontaneously hypertensive rats compared with males

Abstract

A mircoarray analysis was performed to identify novel inflammatory genes that are differentially expressed in the mesenteric arteries of male and female spontaneously hypertensive rats (SHRs). Fractalkine was found to be the inflammatory gene with the greatest differential expression in mesenteric arteries, with the expression being greater in female SHRs compared with males. Greater inflammatory mediators in female SHRs were verified by measuring urinary monocyte chemoattractant protein-1, transforming growth factor-β, and tumor necrosis factor-α (TNF-α) excretion, all of which were greater in female SHRs compared with males. Real-time PCR, Western blot analysis, and ELISA verified greater soluble fractalkine in mesenteric arteries of female SHRs. Consistent with increased fractalkine expression, TNF-α-converting enzyme and TNF-α levels in mesenteric arteries were also greater in female SHRs. We next tested the hypothesis that mesenteric arteries from female SHRs will have greater fractalkine-induced dysfunction. Acetylcholine, sodium nitroprusside, phenylephrine, and KCl concentration-response curves were performed in third-order mesenteric arteries from male and female SHRs pretreated with either vehicle or fractalkine (1 μg/ml). Fractalkine decreased sensitivity to 1) acetylcholine in arteries from male SHRs, 2) phenylephrine in arteries from both sexes, and 3) KCl in arteries from female SHRs. In conclusion, urinary and vascular markers of inflammation are greater in female SHRs compared with males, although blood pressure and cardiovascular risk are less in females.

there is increasing evidence to support a role for inflammation and inflammatory mediators in the development of a number of cardiovascular disorders, including hypertension (4, 27). Spontaneously hypertensive rats (SHRs) are a genetic model of essential hypertension, and male SHRs have a more rapid increase in blood pressure over time compared with female SHRs (32, 33). While a number of factors have been identified that contribute to sex differences in cardiovascular pathologies, to our knowledge, nothing is known regarding potential sex differences in inflammatory mediators in hypertension. This study employed a microarray approach to identify novel inflammatory genes that are differentially expressed in the vasculature of male and female SHRs.

Fractalkine (CX₃CL1, FKN) was identified in microarray studies to be more highly expressed in the mesenteric arterial bed of female SHRs compared with male SHRs. FKN is a unique member of the chemokine superfamily, which exists in both membrane-bound and soluble forms (5). Membrane-bound FKN and its receptor (CX₃CR1) mediate a novel mechanism of leukocyte capture and adhesion under physiological flow, whereas the soluble form possesses potent chemoattractant properties that contribute to the recruitment of monocytes, natural killer (NK) cells, and T lymphocytes in peripheral tissue (9). FKN staining is absent in healthy coronary arteries, although the expression is stimulated by inflammatory mediators, and patients with atherosclerosis, diabetes, and transplant vascular disease have pronounced FKN staining throughout their coronary arteries (38).

The biological roles of FKN are still being investigated, since no obvious defect was found in FKN knockout mice. However, FKN is known to contribute to the development and progression of atherosclerosis in mice (7, 8, 18, 34), and polymorphisms in CXC₃R1 influence the risk of acute coronary events in people (22). This is the first study to examine FKN in a model of essential hypertension; however, patients with pulmonary arterial hypertension have an increase in the fraction of lymphocytes expressing CX₃CR1, plasma-soluble FKN levels, and pulmonary artery endothelial cell FKN mRNA (3).

Based on our microarray data, we hypothesized that inflammatory mediators would be more highly expressed in female SHRs compared with males. Therefore, the first goal of this study was to determine whether there was a sex difference in inflammatory mediators in SHRs. The second goal was to determine the effect of exogenous FKN on vascular function. FKN has been shown to induce vascular dysfunction in aorta from male Wistar rats, although nothing in known regarding the effect of FKN on small artery function, on arteries from females, or on arteries under hypertensive conditions (28). Therefore, we tested the hypothesis that FKN would induce vascular dysfunction in small mesenteric arteries from SHRs. Furthermore, we predict the effect of FKN to be more pronounced in arteries from females.

METHODS

Animals.

Male and female SHRs (Harlan, Indianapolis, IN) were studied at 12 to 13 wk of age. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved and monitored by the Medical College of Georgia Institutional Animal Care and Use Committee. Female rats were selected randomly, and the stage of the estrus cycle was not determined. Rats were housed in temperature- and humidity-controlled and light-cycled quarters and maintained on standard rat chow (Harlan Teklad, 8604). At 13 wk of age, rats were anesthetized with pentobarbital sodium (Nembutal, 65 mg/kg ip; Abbott, North Chicago, IL) and euthanized by exsanguination.

RNA isolation and microarray.

The mesenteric arterial bed was rapidly isolated in ice-cold phosphate-buffered saline and snap frozen in TRIzol. Total RNA was extracted following the homogenization of frozen arterial beds in TRIzol. We performed 14 microarrays, 7 for males and 7 for females. Each microarray was run with RNA pooled from two mesenteric arterial beds, i.e., 14 males and 14 females. RNA was purified by RNeasy minicolumn (Qiagen, Valencia, CA) and then quantified in a spectrophotometer. Preparation of cRNA, hybridization, and scanning of the GeneChip 430 2.0 arrays were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 μg of cRNA were hybridized to a Test2 Array to verify the quality of the cRNA sample. When the cRNA was of sufficient quality, 15 μg of cRNA were hybridized to the Rat Affymetrix U34A GeneChip and scanned. The Affymetrix mouse GeneChip 430 2.0 array was used for hybridization. The Affymetrix GCOS expression analysis software was used to process image data and to generate intensity value for each probe set. Intensity values were compared using a t-test, and the P values were corrected using the Benjamini and Hochberg false-discovery-rate test.

RT-PCR.

Expanded methods are available in the supplemental materials (note: supplemental material may be found with the online version of this article). Briefly, total RNA was isolated and purified as described in RNA isolation and microarray. Gene expression was quantified using real-time PCR (Roche LightCycler). The primers used for real-time PCR experiments were designed using InforMax Vector NTI Advance 9.0 (Invitrogen life sciences software, Frederick, MD, see supplemental material for primer pairs). RNA (2 μg) per sample with a 20 μl total reaction volume was used in all reactions. cDNA was purified using the QIAquick PCR Purification kit (Qiagen), with an additional ethanol washing step added. Standards for each gene were created using conventional PCR and tested for purity and correct size by gel electrophoresis. All gene expression was normalized to GAPDH mRNA levels, and all samples were run in triplicate and averaged.

FKN measurements and Western blot analysis.

The mesenteric bed was isolated and homogenized as previously described (31). FKN was measured in the cytosolic and particulate fractions of the rat mesenteric arterial bed by ELISA according to the manufacturer's instructions (RayBiotech, Norcross, GA). Western blot analysis was performed as previously described (33). Two-color immunoblots were performed using polyclonal primary antibodies to FKN (1:500; Torrey Pines) or CX₃CR1 (1:500; Abcam) in conjunction with a monoclonal antibody to actin (Sigma, St. Louis, MO). Both FKN and CX₃CR1 have a molecular mass of ∼50 kDa.

Immunohistochemical analysis.

Briefly, the mesenteric vascular bed was isolated, fixed in formalin, paraffin embedded, and sectioned at a thickness of 4 μm onto Superfrost plus slides. The slides were incubated in the absence or presence of primary antibody to FKN (1:600; Torrey Pines) or ED-1 (CD58; Serotec, Kidlington, Oxford, UK) in humidity chambers overnight at 4°C, followed by an incubation with peroxidase-conjugated goat anti-mouse IgG (Serotec) for 30 min at room temperature. Specific staining was detected with diaminobenzamidine (DakoCytomation), counterstained with Mayers hematoxylin, and coverslipped.

Vasoreactivity.

A third-order mesenteric artery was isolated and placed in the chamber of a wire myograph (Danish Myo Technology) as previously described (30). Passive tension was set, and there was not a difference in the degree of passive tension in arteries from males and females (see supplemental material). Vessels were equilibrated for 30 min before the viability of the vessel was determined by contracting the vessel with 1 μmol/l phenylephrine (PE) followed by 10 μmol/l acetylcholine (ACh). Drugs were rinsed out and the vessels were reequilibrated for 30 min, and a cumulative concentration-response curve was performed to the endothelium-dependent vasodilator ACh (1 pmol/l–31.6 μmol/l) or the endothelium-independent vasodilator sodium nitroprusside (SNP, 100 pmol/l-31.6 μmol/l, in the presence of 100 μmol/l N^G-nitro-l-arginine) in the absence or presence of FKN (30 min incubation 1 μg/ml; EMD Chemicals, San Diego, CA). FKN did not alter passive tension (see supplemental material). Arteries were preconstricted with PE to 80% maximum constriction, determined when testing artery viability. There was no difference in the level of precontraction between the different rat groups (see supplemental material). Cumulative concentration-response curves were also performed to the vasoconstrictors PE (1 nmol/l–31.6 μmol/l) and KCl (4.7–100 mmol/l) in the absence or presence of FKN. Each agonist concentration was added only after the vessel had reached a plateau from the previous dose, and one curve was obtained from each vessel.

Inflammatory mediator measurements.

An additional set of animals were placed in metabolic cages to allow for 24-h urine collection. Urinary monocyte chemoattractant protein-1 (MCP-1) was measured by ELISA according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ). Transforming growth factor-β (TGF-β) was measured by immunoassay in the urine according to the manufacturer's instructions (Promega, Madison, WI). Tumor necrosis factor-α (TNF-α) was measured by ELISA according to the manufacturer's instructions in urine and mesenteric arterial bed lysates from male and female SHRs (Rat ultraSensitive, Biosource, Camarillo, CA). Tissue was processed as previously described with minor modifications (28). Briefly, the mesenteric arterial bed was isolated and snap frozen. The frozen bed was homogenized in hypotonic buffer, containing (in mM) 20 HEPES (pH 7.4), 10 NaCl, 1 vanadate, 10 NaF, and 10 EDTA, in the presence of protease inhibitors, containing 1 mg/ml PMSF, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A, at a ratio of 7:1 (wt:vol). The homogenate was centrifuged at 15,000 g at 4°C for 20 min, and TNF-α was measured in the soluble extract and normalized to protein. Protein concentrations were determined by standard Bradford assay with bovine serum albumin as the standard.

Data analysis.

All other data are expressed as means ± SE. Microarray data, Western blot data, and inflammatory measurements were compared by Student's t-test (OriginLab, Northampton, MA). Negative log of the EC₅₀ (pD₂) values were calculated by nonlinear regression of the sigmoidal dose-response curve, and the pD₂ values were compared by a one-way ANOVA (GraphPad Prism, San Diego, CA).

RESULTS

Gene expression.

When hybridized onto an Affymetrix GeneChip, ∼3,500 genes (40% of genes represented on the array) were present in mesenteric arteries. A majority of the genes had similar gene expression profiles in the mesenteric arterial bed of male and female SHRs. To be considered differentially expressed, a gene had to be present on at least 11 of the 14 arrays (7 arrays for males and 7 for females). Once this criterion had been met, male and female group comparisons were made using t-tests; P < 0.01 was considered significantly different. There were 125 genes differentially expressed between the mesenteric arterial bed of males and females. Of these genes, 26 were more highly expressed (1.5-fold or greater) in arteries from males (Table 1) and 79 had greater expression (1.5-fold or greater) in arteries from females ( Table 2). See supplemental material for genes with a differential expression of 1- to 1.5-fold. Five of the genes with the greatest fold change in expression in mesenteric arteries from male and female SHRs were examined by real-time PCR to verify the microarray results (Fig. 1). We verified that four of the five genes examined, rat PERIOD2 protein, enhancer-of-split and hairy-related protein 1, FKN, and decorin, were differentially expressed.

Table 1.

Genes expressed 1.5-fold and greater in the mesenteric arterial bed of male SHRs compared with females

Accession No.	Fold Difference	P Value	Gene Description
J03179	5.66	<0.001	D-binding protein
AF009329	4.26	<0.001	Enhancer-of-split and hairy-related protein 1 (SHARP-1)
AB016532	3.79	<0.001	rat PERIOD2 protein (rPER2)
U20796	1.92	0.001	Nuclear receptor Rev-ErbA-β
U08290	1.86	0.007	Neuronatin-α
D31838	1.84	0.001	Wee1 tyrosine kinase
M26125	1.79	0.001	Epoxide hydrolase
AA893244	1.76	0.003	3′-Phosphoadenosine 5′-phosphosulfate synthase 2
U24652	1.75	0.003	Lnk1
M24067	1.74	0.004	Plasminogen activator inhibitor-1 (PAI-1)
U47031	1.74	0.005	P2x4 ATP receptor
H31665	1.71	0.010	Hypoxia-induced gene 1
M91652	1.59	0.008	Glutamine synthetase (glnA)
AF019043	1.57	0.006	Dynamin-like protein (DLP1)
M22400	1.56	<0.001	Developmentally regulated intestinal protein (OCI-5)
U53855	1.53	0.003	Prostacyclin synthase
AA891916	1.51	0.003	Membrane interacting protein of RGS16

Table 2.

Genes expressed 1.5-fold and greater in the mesenteric arterial bed of female SHRs compared with males

Accession No.	Fold difference	P Value	Gene Description
AF030358	2.88	0.003	Chemokine CX3C
AI102044	2.73	0.006	Mitochondrial protein
M76767	2.51	0.009	Fatty acid synthase
X59859	2.33	<0.001	Decorin
AI638985	2.26	<0.001	Ligand-dependent nuclear receptor corepressor-like (Lcorl), transcript variant 1
AI639155	2.22	0.001	Protein-l-isoaspartate (d-aspartate) O-methyltransferase domain
AA859612	2.12	<0.001	Mitochondrial protein
AI009682	2.05	0.001	Aspartyl-tRNA synthetase
K00750 exon no. 2-3	2.04	0.002	Cytochrome c nuclear-encoded mitochondrial gene and flanks
U08976	2.04	0.006	Peroxisomal enoyl hydratase-like protein
AI171506	2.00	0.007	Malic enzyme (MAL)
AI236721	1.95	<0.001	Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, γ-polypeptide
S59892	1.91	0.002	La = autoantigen SS-B/La
AI008815	1.90	<0.001	Cytochrome c, somatic
AA875059	1.82	0.001	Mus musculus 13 days embryo forelimb cDNA
AA894282	1.78	0.001	cDNA clone MGC:93828
AA874873	1.75	0.004	Mus musculus adult male cecum cDNA, RIKEN
U67082	1.74	0.005	KRAB-zinc finger protein KZF-1
S59893	1.71	0.007	La = autoantigen SS-B/La
AJ000347	1.67	0.001	3(2),5 -bisphosphate nucleotidase
AI639410	1.66	0.002	Signal-transducing adaptor molecule (SH3 domain and ITAM motif) 2 (Stam2)
AI014135	1.66	0.009	Mitochondrial protein
AI639324	1.64	0.004	F-box protein 38
AJ012603 UTR no. 1	1.64	<0.001	TNF-α-converting enzyme (TACE)
M31174	1.63	0.004	c-erbA-α-2-related protein
AA859829	1.62	0.001	Macrophage erythroblast attacher
AI639151	1.61	0.002	Pinin
D49708	1.61	0.002	RNA-binding protein (transformer-2-like)
AA866426	1.60	0.002	Mus musculus BAC clone RP23-77G5 from 18
AF016387	1.60	0.005	Retinoid-X receptor-γ (RXRγ)
U63839	1.60	0.007	Nucleoporin p58
U97146	1.58	0.006	Calcium-independent phospholipase A2
X17053cds	1.58	0.001	Immediate-early serum-responsive JE gene
AA818983	1.57	0.005	Diacylglycerol kinase, β
AA818240	1.55	0.004	Nucleoporin 153
AA892137	1.55	0.007	CREBBP/EP300 inhibitory protein 1 (predicted)

Fig. 1.

Differential RNA expression by microarray analysis (A) and real-time PCR (B) in mesenteric arteries of male and female spontaneously hypertensive rats (SHRs). Real-time PCR expression levels are normalized to GAPDH levels. Values for males were set to 1, and values for females are expressed as fold change relative to males. FKN, fractalkine. *P < 0.05, significant difference from males. Numbers in parentheses refer to n values.

Interestingly, a number of the genes identified to be differentially expressed are involved in inflammation, including FKN, decorin, TACE, macrophage erythroblast attacher, and rat mast cell protease 7. Expressions of all of these genes were found to be greater in the mesenteric arterial bed of female SHRs compared with males.

Urinary inflammatory measurements are greater in female SHRs.

To assess whether the sex difference in inflammatory mediators is a general phenomenon, we assayed MCP-1, TNF-α, and TGF-β levels in the urine of age-matched male and female SHRs. MCP-1 excretion was significantly lower in male SHRs compared with females (2.2 ± 0.5 vs. 5.3 ± 0.4 ng/day, respectively, P < 0.05; n = 8). The excretion of both TNF-α, and TGF-β were below detection in male SHRs. In female SHRs, the excretion of TNF-α was 136 ± 20 pg/day (n = 6) and TGF-β excretion was 358 ± 62 ng/day (n = 8).

FKN expression is higher in arteries from female SHRs.

The remainder of this study focused on FKN, the inflammatory gene identified to have the greatest fold change in expression in the mesenteric arteries of male and female SHRs. Protein levels were determined for both FKN and the FKN receptor. Since FKN can exist in soluble and membrane-anchored forms, Western blot analysis was performed on mesenteric arterial tissue separated into soluble and particulate fractions (Fig. 2). There was significantly greater FKN in the soluble fraction of mesenteric arteries from female SHRs compared with males (Fig. 2A, P < 0.05). Particulate FKN expression and FKN receptor expression were comparable in arteries from males and females (Fig. 2C). Significantly greater FKN in the soluble fraction of mesenteric arteries from female SHRs was quantified and verified by ELISA (Fig. 2B, P < 0.005). To determine whether greater levels of FKN were associated with the female sex or the presence of hypertension, FKN levels were quantified in the mesenteric arterial bed of age-matched male and female Wistar-Kyoto (WKY) rats. FKN levels were comparable in both the soluble (4.1 ± 0.6 vs. 3.3 ± 0.7 ng/mg, n = 8) and the particulate fractions (0.75 ± 0.06 vs. 0.90 ± 0.16 ng/mg, n = 8) of mesenteric arteries from male and female WKY rats. Plasma FKN levels were also measured in male and female WKY and SHRs to determine whether there is a systemic increase in FKN in female SHRs or whether the observed increase is tissue specific. Plasma FKN levels were greater in SHRs (male, 242 ± 9; and female, 214 ± 11 pg/ml) than in WKY (male, 176 ± 11; and female, 138 ± 7 pg/ml); however, there were no apparent sex differences.

Fig. 2.

Western blot analysis of FKN ligand (A) and receptor (Cx₃CR1; C) in the soluble (S) and particulate (P) fraction of mesenteric arteries from male and female SHRs. The first lane in the representative Western blot is the molecular weight marker (MWM). B: quantification of protein levels of FKN by ELISA. *P < 0.05, a significantly greater compared with male S. Numbers in parentheses refer to n values.

FKN localization in mesenteric arteries of SHRs.

An immunohistochemical analysis was performed to examine the cellular localization of FKN and macrophage infiltration. FKN was found to be prominently expressed in the vascular endothelium, with some medial staining (Fig. 3, A and B). Since FKN expression has been shown to be associated with an increase in inflammatory cell adhesion and migration to the vasculature, we examined macrophage infiltration to determine whether some of the medial cell staining of FKN was found in association with macrophages. In fact, there were few macrophages associated with mesenteric arteries from either male or female SHRs, and there was no detectable infiltration in the medial layer (Fig. 3, C and D).

Fig. 3.

FKN and macrophage staining in third-order mesenteric arteries from male (A and C) and female (B and D) SHRs. Arrows indicate ED-1⁺ cells. FKN images were taken using a ×1,000 objective; macrophage images were taken using a ×200 objective.

TACE and TNF-α levels are greater in mesenteric arteries from female SHRs.

Inflammatory agents and other chemokines stimulate FKN expression, and soluble FKN is formed by the cleavage of membrane-bound FKN by TACE. TACE was identified in the microarray analysis to be more highly expressed in mesenteric arteries of females compared with males, consistent with females having greater soluble FKN expression (Fig. 4B). TNF-α stimulates FKN expression in cultured endothelial cells (1). Therefore, we examined whether a greater FKN expression in arteries from females was also associated with a greater TNF-α. Indeed, the lysates of mesenteric arteries from female SHRs had greater levels of TNF-α in female SHRs compared with male SHRs (Fig. 4).

Fig. 4.

TNF-α-converting enzyme (TACE) mRNA differential gene expression (A) and TNF-α protein levels (B) in the mesenteric arterial bed from male and female SHRs. *P < 0.01, significantly greater compared with male SHRs. Numbers in parentheses refer to n values.

FKN alters vascular function.

FKN has been shown to induce vascular dysfunction in large conduit arteries (28). Therefore, we first examined the ability of acute incubation with FKN to modulate vasodilator reactivity. Dilator responses to the endothelium-dependent vasodilator, ACh, and the endothelium-independent dilator, SNP, were comparable in small mesenteric arteries from male and female SHRs under control conditions (Fig. 5; for all pD₂ and maximum response values, see supplemental material). Following a 30-min incubation with FKN, the sensitivity to ACh was significantly decreased in arteries from male SHRs compared with all other treatment groups (P < 0.01), although the maximal relaxation was maintained. In contrast, neither sensitivity nor maximal relaxation to ACh was altered in arteries from females (Fig. 5A). To assess whether the decrease in sensitivity to ACh following FKN incubation in arteries from male SHRs was dependent on the vascular endothelium, cumulative concentration-response curves were performed to the endothelium-independent nitric oxide donor SNP (Fig. 5B). There were no significant differences in either sex following FKN incubation in vasorelaxation to SNP, although there was a trend for FKN to decrease sensitivity to SNP in arteries from male SHRs.

Fig. 5.

ACh (n = 7–9; A) and sodium nitroprusside (SNP; n = 4–6; B) concentration-response curves in mesenteric arteries from male and female SHRs in the absence and presence of FKN (1 μg/ml, 30 min). PE, phenylephrine. See supplemental material for all negative log of the EC₅₀ (pD₂) values. *P < 0.05, significant decrease in sensitivity compared with control male.

Contractile responses were next examined to the adrenergic agonist PE and the nonspecific constrictor KCl. Vasoconstriction profiles to both agents were comparable in small mesenteric arteries from male and female SHRs under control conditions (Fig. 6; for all pD₂ and maximum response values, see supplemental material). Following a 30-min incubation with FKN, the sensitivity to PE was significantly decreased in arteries from both male and female SHRs, although there was no change in maximal contraction in either sex (Fig. 6A). To assess whether the decrease in sensitivity to PE following FKN incubation was indicative of a generalized impairment of smooth muscle contractility, cumulative concentration-response curves were performed to KCl (Fig. 6B). FKN incubation has no effect on KCl responses in arteries from male SHRs. In arteries from female SHRs, however, FKN incubation resulted in a significant decrease in sensitivity and maximum response to KCl.

Fig. 6.

PE (n = 4–13; A) and KCl (n = 6–11; B) concentration-response curves in mesenteric arteries from male and female SHRs in the absence and presence of FKN (1 μg/ml, 30 min). See supplemental material for all pD₂ values. *P < 0.05, significant decrease in sensitivity compared with control male. +P < 0.05, significant decrease in sensitivity compared with control female.

DISCUSSION

There is increasing interest in the role of inflammation and inflammatory mediators in the pathogenesis of cardiovascular disorders, including hypertension. Nothing is known, however, regarding sex differences in inflammatory mediators in hypertension. Microarray studies were performed with the intent of identifying novel inflammatory genes that are differentially expressed in the vasculature of male and female SHRs. We identified five proinflammatory genes that were differentially expressed in the mesenteric arterial bed of male and female SHRs, with the expression of all five of these genes being greater in female SHRs compared with males. This result was supported by urinary and vascular measurements of proinflammatory mediators. The main finding of this study was that FKN was the gene with the highest differential expression in the mesenteric arterial bed with soluble FKN levels being greater in female SHRs. We confirmed FKN localization in the endothelium from both male and female SHRs. The ability of FKN to modulate vascular function was tested by incubating isolated arteries with FKN and examining vascular function. Exogenous FKN resulted in endothelial dysfunction in arteries from male SHRs and in vascular smooth muscle dysfunction in arteries from female SHRs. It is interesting to note that despite female SHRs having higher levels of inflammatory mediators in urine and in the vasculature, they maintain a lower blood pressure compared with age-matched male SHRs (33). Thus the blood pressure and the expression of inflammatory mediators do not appear to correlate in SHRs.

There is increasing evidence to support the role for inflammation in the development of hypertension (4, 25, 27). Vascular inflammation has been demonstrated in both hypertensive patients and experimental animals (11, 12, 14); however, much less is known regarding the influence of sex of the animal on inflammatory mediators in cardiovascular disease. This is the first study to measure inflammatory mediators in a model of essential hypertension in males and females, and for all inflammatory mediators that were assessed, the levels were greater in females compared with males. This is consistent with clinical data showing that women have increased immune activity and greater autoantibody production compared with men and with experimental data reporting that females have increased rejection of transplant tissue compared with males (16, 17, 29). The question remains, however, as to whether increased inflammatory mediators in female SHRs equate with increased levels of tissue inflammation. To address this point, we assessed the macrophage infiltration in mesenteric arteries and found that despite greater FKN in female SHRs, macrophage infiltration into arteries was minimal in both sexes. These data raise the possibility that SHRs have compensatory mechanisms that modulate the inflammatory response, and this response is greater in females compared with males.

In this study, we focused on FKN, the gene identified to have the greatest differential expression in the mesenteric arteries of female and male SHRs. FKN exists in two forms, soluble and membrane anchored, both with distinct functions. We determined that soluble FKN is more highly expressed in the mesenteric arterial bed of female SHRs than in male SHRs. Plasma levels of FKN were also measured in male and female WKY and SHRs, and although FKN levels were greater in SHRs compared with WKY rats, there were no sex differences in FKN expression. These data suggest that FKN levels increase with hypertension in a tissue-specific manner. One of the primary stimulants of FKN expression is TNF-α. TNF-α increases FKN expression in cultured endothelial cells, arterial endothelial cells, and cultured rat aortic smooth muscle cells (1, 6). Both TNF-α and TACE, the enzyme that cleaves membrane-bound FKN (10, 35), were more highly expressed in arteries of female SHRs compared with males. Interestingly, the serum levels of TNF-α are elevated in aged, estrogen-deficient rats, resulting in vascular dysfunction of mesenteric arteries (2). Therefore, we speculate that in females, the presence of cardiovascular risk factors increases TNF-α and TACE, which may drive the greater FKN expression in female SHRs. Future studies are needed to address this hypothesis.

Soluble FKN has potent chemoattractant properties contributing to the recruitment of monocytes, NK cells, and T lymphocytes in peripheral tissue. Membrane-bound FKN expression is found predominately at the lumenal surface of injured vascular endothelial cells and directly mediates cell-cell interactions to increase the adhesion and migration of inflammatory cells (5, 36). Consistent with this finding, we found distinct endothelial FKN staining in mesenteric arteries from SHRs. Not surprisingly, FKN has been shown to contribute to the development of arterial injury and atherosclerotic lesions (7, 18, 19). With regards to sex, FKN deficiency decreases the lesion area in apolipoprotein E and low-density lipoprotein receptor knockout mice, and this effect is more pronounced in female mice compared with males (34). The FKN receptor, CX₃CR1, is expressed on monocytes/macrophages, NK cells, and some CD8⁺ T cells (36), and in mice, the blockade of the FKN receptor using CX₃CR1 antibody decreases kidney macrophage infiltration following acute renal failure and attenuates the development of renal injury (24). Although, historically, soluble FKN has been shown to promote inflammatory cell adhesion and migration, recently NK cell adhesion to FKN immobilized on glass slides or to cultured endothelial cells expressing FKN can be inhibited by soluble FKN, and in endothelial cells, soluble FKN inhibits NK cell-mediated cell lysis (40). Therefore, under certain conditions, increased soluble FKN may serve as a mechanism to limit inflammatory cell adhesion and migration. Soluble FKN has also been shown to antagonize the chemoattractant effect of MCP-1 on cultured monocytes (37) and to suppress NK cell adhesion and cytolysis of FKN-transfected endothelial cells (39). It is possible, then, that greater soluble FKN in female SHRs is a compensatory mechanism to inhibit cell adhesion and to limit the inflammatory response. This explanation would account for the apparent discrepancy between increased levels of inflammatory markers in female SHRs, yet lower levels of blood pressure and cardiovascular damage compared with those in male SHRs. Future studies using FKN neutralizing antibodies are needed to determine the physiological consequence of greater soluble FKN expression in female SHRs.

This is the first study to examine the effects of acute FKN treatment on vascular reactivity in small arteries; however, exogenous FKN has been shown to blunt ACh-induced relaxation in the thoracic aorta from male Wistar rats (28). Incubation with FKN resulted in endothelial dysfunction in small mesenteric arteries from male SHRs, as evidenced by a decreased in sensitivity to ACh and a trend for a decrease in sensitivity to SNP-induced relaxation and vascular smooth muscle dysfunction in arteries from female SHRs, as evidenced by a decreased sensitivity to PE and KCl. Therefore, our data suggest that an incubation with FKN results in a sex difference in the susceptibility of the vasculature to FKN-induced damage. The mechanisms responsible for the sex differences in response to FKN incubation are not known. There may be sex differences in the CX₃CR1. Although there was no difference in the protein expression of the FKN receptor (CX₃CR1) in the mesenteric arterial bed, a sex difference in CX₃CR1 polymorphisms may contribute to sex differences in FKN responses since polymorphisms in the CX₃CR1 have been shown to influence the development of cardiovascular disorders (19–21). In contrast to our finding, FKN blunted relaxation to the nitric oxide donor diethylammonium(2)-1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate in the aorta from male Wistar rats; however, the duration of the incubation with FKN was 2 h compared with our 30-min incubation (28). Additionally, conduit arteries may respond differently from small arteries, and the blood pressure status of the rats may alter the vascular reactivity responses. It should be noted that all biochemical measurements were performed using the entire mesenteric arterial bed, which includes both large and small arteries, whereas the functional studies were performed in isolated third-order arteries.

The experiments were also performed to determine the effect of sex on vascular responses in small mesenteric arteries. There is not a consensus in the literature as to whether or not sex influences ACh-induced vasorelaxation in SHRs. ACh-induced relaxation in PE-precontracted aorta has been shown to be greater in 16-wk-old female SHRs compared with age-matched males and comparable with aorta from 11-wk-old male and female SHRs (13, 39). Similarly, in the superior mesenteric artery, ACh-induced relaxation is comparable in arteries from 12-wk-old male and female SHRs, and either are not different in KCl-precontracted arteries or greater in PE-precontracted arteries from 20-wk-old females compared with males (11a, 23). We report that there is no difference in ACh-induced relaxation in small, third-order mesenteric arteries from male and female SHRs. This is consistent with reports in larger arteries from similarly aged animals when precontracted with PE.

In conclusion, vascular and urinary inflammatory mediators are greater in female SHRs compared with males. This is the first study to compare inflammatory mediators between males and females under hypertensive conditions. Despite females having greater levels of inflammatory mediators, females have a lower blood pressure and less overall cardiovascular risk compared with their male counterparts. It is possible that in conjunction with greater inflammatory mediators in females, there is also an upregulation of anti-inflammatory pathways that limit inflammatory-induced cardiovascular dysfunction.

GRANTS

This study was funded by National Institutes of Health Grants HL-69999 (to J. S. Pollock), AG-024616 (J. C. Sullivan), and 5U24-DK-58778 (J.-X. She). J. Pollock is an Established Investigator of the American Heart Association. J. Sullivan is a recipient of a Scientist Development Grant from the American Heart Association.