Hypertension induces gonadal macrophage imbalance, inflammation, lymphangiogenesis, and dysfunction

Abstract

Hypertension (HTN) is associated with gonadal dysfunction and impaired reproductive health in both men and women. An imbalance in the systemic and renal proinflammatory (M1)/anti-inflammatory (M2) macrophage ratio, increased inflammation, and inflammation-associated lymphangiogenesis have been observed in animals with HTN. However, the impact of HTN on gonadal macrophages, inflammation, and lymphatics remains obscure. We hypothesized that salt-sensitive HTN (SSHTN) and HTN alters gonadal macrophage polarization, which is associated with inflammation, inflammation-associated lymphangiogenesis, and reproductive dysfunction. Flow cytometry analyses revealed a significant increase in M1 macrophages in the testes of SSHTN and nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced HTN (LHTN) mice, with a concurrent decrease in M2 macrophages in SSHTN mice yet an increase in M2 macrophages in LHTN mice. Ovaries from SSHTN mice exhibited an increase in M1 and a decrease in M2 macrophages, while ovaries from LHTN mice had a significant increase in M2 and a decrease in M1 macrophages. Gene expression patterns of proinflammatory cytokines revealed gonadal inflammation in all hypertensive mice. Increased lymphatic vessel density in the gonads of both male and female hypertensive mice was confirmed by immunofluorescence staining for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). HTN adversely affected the expression pattern of steroidogenic enzymes, hormone receptors, and secretory proteins in both the testes and ovaries. In line with these results, male hypertensive mice also presented with decreased sperm concentration, and increased percentage of sperm with abnormal morphology, damaged acrosome, and nonfunctional mitochondrial activity. These data demonstrate that HTN alters gonadal macrophage polarization, which is associated with gonadal inflammation, inflammation-associated lymphangiogenesis, and dysfunction.

Keywords: Hypertension; Lymphatics; Macrophages; Ovaries; Testes.

Figure 1.. SSHTN mice had increased M1 macrophages, decreased M2 macrophages, and inflammation in the gonads

SBP measures in (A) male and (B) female control and mice administered L-NAME in the drinking water for 2 weeks, then 2 weeks of tap water washout, and then 3 weeks of 4% salt diet (SSHTN) (n=6 in both males and females). Macrophage (M1 and M2) populations expressed as percentage of CD11b⁺F4/80⁺ cells in (C) testes and (D) ovaries from control and SSHTN mice, as determined by flow cytometry (n=8 in both males and females). Gene expression of proinflammatory mediators in (E) testes and (F) ovaries from control and SSHTN mice (n=6 in both males and females). Results are expressed as mean ± SEM, and statistical analysis consisted of a Student’s t test. *P<0.05 vs control.

Figure 2.. SSHTN increased lymphatic vessel density in the gonads

Lymphatic vessel density in (A) testes and (B) ovaries from control and SSHTN mice as determined by LYVE-1+ pixels relative to DAPI per field. Representative images of LYVE-1 immunofluorescence in testis (scale bar = 100 μm) and ovary (scale bar = 200 μm) sections (n=6 in both males and females). Green: LYVE-1; Red: CD31; Blue: DAPI. Gene expression of lymphatic vessel markers in (C) testes and (D) ovaries from the control and SSHTN mice (n=6 in both males and females). Results are expressed as mean ± SEM, and statistical analyses consisted of Student’s t test. *P<0.05 vs control. Representative 3-D model of confocal images of clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) cleared testis immunostained with LYVE-1 from (E) control and (F) SSHTN mice, n=3. Representative 3-D model of confocal images of clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) cleared ovary immunostained with LYVE-1 from (G) the control and (H) SSHTN mice, n=3. Scale bars = 1000 μm (testis) and 500 μm (ovary).

Figure 3.. SSHTN mice exhibited gonadal dysfunction

Gene expression of steroidogenic pathway genes and hormone receptors in (A) testes from control and mice-administered L-NAME in the drinking water for 2 weeks, then 2 weeks of washout, and then a subsequent 3 weeks of 4% salt diet (SSHTN), n=6. Gene expression of secretory proteins and tight junction proteins in (B) testes from control and SSHTN mice, n=6. Sperm functional tests in control and SSHTN mice demonstrate (C) decreased sperm concentration, n=8, (D) increased percentage of sperm with abnormal morphology, n=8, (E) increased percentage of sperm with damaged acrosome, n=8, and (F) increased number of sperm with nonfunctional mitochondrial activity n=8. Representative images of (i) intact and (ii) damaged acrosome integrity were assessed using FITC-PNA that binds exclusively to the outer membrane of the acrosome. Scale bars = 10 μm. Florescent dye Rh123 (Rhodamine123) distinguishes (iii) functional and (iv) nonfunctional sperm mitochondria as only live cells can retain the stain after washing. Scale bars = 20 μm. Expression of steroidogenic pathway genes, hormone receptors, and secretory proteins in (G) ovaries from control and SSHTN mice, n=6. Results are expressed as mean ± SEM, and statistical analysis consisted of a Student’s t test. *P<0.05 vs control.

Figure 4.. LHTN was associated with altered macrophage polarization and inflammation in the gonads

SBP measures in (A) male and (B) female control and mice administered L-NAME in the drinking water for 3 weeks (LHTN) (n=6 in both males and females). Macrophage (M1 and M2) populations expressed as percentage of CD11b⁺F4/80⁺ cells in (C) testes and (D) ovaries from control and LHTN mice, as measured by flow cytometry (n=8 in both males and females). Gene expression of proinflammatory mediators in (E) testes and (F) ovaries from control and LHTN mice (n=6 in both males and females). Results are expressed as mean ± SEM, and statistical analyses consisted of Student’s t test. *P<0.05 vs control.

Figure 5.. LHTN increased lymphatic vessel density in the gonads

Lymphatic vessel density in (A) testes and (B) ovaries from the control and LHTN mice as determined by LYVE-1+ pixels relative to DAPI per field (n=6 in both males and females). Representative images of LYVE-1 immunofluorescence in testis (scale bar = 100 μm) and ovary (scale bar = 200 μm) sections. Green: LYVE-1; Red: CD31; Blue: DAPI. Gene expression of lymphatic vessel markers in (C) testes and (D) ovaries from the control and LHTN mice (n=6 in both males and females). Results are expressed as mean ± SEM, and statistical analyses consisted of Student’s t test. *P<0.05 vs control. Representative 3-D model of confocal images of clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) cleared testis immunostained with LYVE-1 from (E) the control and (F) LHTN mice, n=3. Representative 3-D model of confocal images of clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) cleared ovary immunostained with LYVE-1 from (G) the control and (H) LHTN mice, n=3. Scale bars = 1000 μm (testis) and 500 μm (ovary).

Figure 6.. LHTN mice exhibited gonadal dysfunction

Gene expression of steroidogenic pathway genes and hormone receptors in (A) testes from the control and mice administered L-NAME in the drinking water for 3 weeks (LHTN), n=6. Gene expression of secretory proteins and tight junction proteins in (B) testes from the control and LHTN mice, n=6. Sperm functional tests in the control and LHTN mice demonstrate (C) decreased sperm concentration, n=8, (D) increased percentage of sperm with abnormal morphology, n=8, (E) increased percentage of sperm with damaged acrosome, n=8, and (F) increased number of sperm with nonfunctional mitochondrial activity, n=8. Representative images of (i) intact and (ii) damaged acrosome integrity were assessed using FITC-PNA that binds exclusively to the outer membrane of the acrosome. Scale bars = 10 μm. Florescent dye Rh123 (Rhodamine123) distinguishes (iii) nonfunctional and (iv) functional sperm mitochondria as only live cells can retain the stain after washing. Scale bars = 20 μm Expression of steroidogenic pathway genes, hormone receptors, and secretory proteins in (G) ovaries from control and LHTN mice, n=6. Results are expressed as mean ± SEM, and statistical analysis consisted of a Student’s t test. *P<0.05 vs control.