Notes on Aging and Endothelin B Receptor (ETBR) Expression in Men

Introduction

• Topic: Impact of aging on the cardiovascular system with a focus on endothelial ETB receptor (ETBR) protein expression in men. Study builds on prior work in women showing age-related ETBR reduction contributing to endothelial dysfunction.

• Key question: Is ETBR protein expression reduced with aging in men, as seen in women, or is it preserved?

• Background concepts:

  • Endothelin-1 (ET-1) is a potent vasoconstrictor produced mainly by endothelial cells.

  • ET-1 signals through two receptors: ETAR and ETBR.

  • ETAR is primarily vasoconstrictive on vascular smooth muscle; ETBR exists on both endothelial cells (vasodilation) and smooth muscle cells (vasoconstriction depending on location).

  • Aging is associated with endothelial dysfunction and increased ET-1 activity; prior data show higher ET-1 in older men and greater endothelial ET-1 in older vs younger men, linked to reduced flow-mediated dilation (FMD).

• Hypotheses tested:

  • Primary: ETBR protein expression is lower in older men (OM) than in younger men (YM).

  • Secondary: ETBR protein expression would correlate positively with FMD.

• Study design overview:

  • Cross-sectional comparison of ETBR expression in venous endothelial cells and endothelial function by FMD in YM vs OM.

  • Endothelial cells harvested from antecubital vein; ETBR expression measured by immunocytochemistry; normalized to HUVECs as control for staining variability; FMD measured per established guidelines.

  • Subset analyses included correlations with testosterone, cholesterol, and glucose.

Methods

• Participants

  • YM: $n=17, ext{ age }=24 ext{ ± } 5 ext{ yr}$; BMI $24 ext{ ± } 2 ext{ kg/m}^2$; restated elsewhere as needed.

  • OM: $n=14, ext{ age }=60 ext{ ± } 6 ext{ yr}$; BMI $26 ext{ ± } 3 ext{ kg/m}^2$.

  • Exclusion: cardiovascular, metabolic, neurologic, or endocrine disease; hypertensive or obese individuals excluded; tobacco use within 6 months excluded.

  • Exercise: 8 of 14 OM reported regular exercise; 14 of 17 YM reported regular exercise.

• Experimental Protocol

  • Pre-test: fasting state for subset; standardizations for recent activity, caffeine, alcohol, and meals (24 h, 12 h, and 4 h as applicable).

  • Visit procedures: supine rest ~20 min; antecubital venous blood draw for endothelial cell harvesting; ~30 min later, FMD measurement of brachial artery via ultrasound.

  • FMD assessment: performed with GE Logiq e system; analyzed with automated edge-detection software (QUIPU).

  • Endothelial cell collection: J-wire method through 18-gauge catheter into antecubital vein (2–4 cm).

  • Cell processing: cells dissociated, fixed in 4% paraformaldehyde, mounted on coverslips; randomized blinding for staining/analysis.

• Immunostaining and imaging

  • Primary ETBR antibody (1:250) and VE-cadherin (1:100) to identify endothelial cells; secondary antibodies (donkey anti-mouse Alexa Fluor 594, donkey anti-rabbit Alexa Fluor 488) used; nucleus stained with DAPI.

  • Coverslips mounted with ProLong Diamond; imaging with ZEISS Axio Imager A2; fixed exposure settings.

  • Quantification: fluorescence intensity measured in ~30 cells per participant and averaged.

  • Normalization: ETBR intensity normalized to ETBR expression in human umbilical vein endothelial cells (HUVECs) to account for staining variability; expressed as ETBR:HUVEC ratio in arbitrary units (AU).

• Blood analyses

  • Serum total testosterone measured in duplicate by ELISA.

  • Subset analyses for cholesterol and blood glucose in 13 OM and 8 YM.

• Statistical analyses

  • Baseline characteristics, ETBR expression, and FMD compared by unpaired two-tailed t-tests (OM vs YM).

  • Pearson correlations explored relationships between ETBR expression, FMD, testosterone, cholesterol, and glucose.

  • Data presented as means ± SD.

Participant characteristics and endothelium function

• Age difference between groups by design: OM significantly older than YM (P < 0.001).

• Cardiometabolic factors (age-related changes observed):

  • Total cholesterol: higher in OM than YM (P < 0.001).

  • LDL: higher in OM than YM (P < 0.001).

  • HDL: higher in OM than YM (P = 0.05).

  • Fasting glucose: higher in OM than YM (P < 0.01).

  • BMI: tendency for higher BMI in OM (P = 0.07).

• Blood pressure: SBP similar (OM: $123 ext{ ± } 11 ext{ mmHg}$ vs YM: $122 ext{ ± } 10 ext{ mmHg}$); DBP higher in OM (OM: $77 ext{ ± } 7 ext{ mmHg}$ vs YM: $70 ext{ ± } 6 ext{ mmHg}$; P < 0.01).

• Hormone levels: Total testosterone lower in OM (OM: $6.28 ext{ ± } 4.21 ext{ ng/mL}$) vs YM (YM: $9.10 ext{ ± } 2.68 ext{ ng/mL}$); P = 0.03.

• Endothelial function (FMD):

  • OM: 3.85 ext{ ± } 1.51 ext{%}; YM: 6.40 ext{ ± } 2.68 ext{%}; P < 0.01.

• ETBR protein expression in endothelial cells

  • OM: $0.39 ext{ ± } 0.17 ext{ AU}$; YM: $0.42 ext{ ± } 0.17 ext{ AU}$; P = 0.66 (no age-related difference).

• Data nuances:

  • Some data points exclude certain participants due to unusable images or analyzability; ETBR expression calculated using available samples (OM n ≈ 12, YM n ≈ 15 for ETBR expression).

ETBR expression and its relation to FMD and metabolic factors

• Primary finding: ETBR protein expression did not differ between OM and YM despite lower FMD in OM, indicating preserved endothelial ETBR with aging in men.

• Correlations:

  • ETBR expression vs FMD: trend toward inverse relation (lower ETBR expression associated with higher FMD), but overall non-significant (P ≈ 0.058; r ≈ -0.39).

  • ETBR expression vs fasting glucose: negative correlation (higher ETBR expression associated with lower glucose); reported as r ≈ -0.49; P ≈ 0.04.

  • ETBR expression not correlated with BMI, exercise frequency, cholesterol measures, or total testosterone (NS).

• Comparative context with prior female data:

  • In postmenopausal women, ETBR expression was reduced vs young women, and ETBR correlated positively with FMD, suggesting a potential ETBR-mediated contribution to endothelial aging in women.

• Notable observations on testosterone mediation in aging men:

  • Some prior work suggests testosterone modulates vasculature and ET-1 signaling; this study explored a potential link between testosterone and ETBR expression but found no significant correlation in men as a group.

  • Exploratory median split in OM by testosterone level suggested higher testosterone within OM may be associated with higher FMD, consistent with testosterone potentially mediating vascular aging effects; however, ETBR expression did not differ by testosterone level in OM (0.37 ± 0.14 AU vs 0.42 ± 0.20 AU; P = 0.63).

  • Other studies indicate testosterone can influence ET-1 production and vasoconstriction, so interactions between androgens, ET-1 system, and ET receptors may exist, warranting further study.

• Exercise considerations:

  • Exercise frequency did not show a clear association with FMD or ETBR expression in this sample, though only 30% were sedentary; larger, quantitatively characterized studies are needed to determine exercise’s impact on ETBR expression/function in men and women.

Figures and their interpretations

• Figure 1A: FMD (%) is lower in OM (n = 13) than YM (n = 14): demonstrate age-related endothelial function decline.

  • OM: ext{FMD} = 3.85 ext{ ± } 1.51 ext{%}; YM: 6.40 ext{ ± } 2.68 ext{%}; P < 0.01.

• Figure 1B: ETBR protein expression in venous endothelial cells (AU) from YM and OM.

  • YM: $0.42 ext{ ± } 0.17 ext{ AU}$; OM: $0.39 ext{ ± } 0.17 ext{ AU}$; P = 0.66.

  • Representative images show ETBR staining in YM vs OM, with VE-Cad as endothelial marker and DAPI for nuclei.

• Figure 1C: Correlation between ETBR expression (AU) and FMD (%).

  • Reported as a trend toward inverse relation (r ≈ -0.39; P ≈ 0.058).

• Figure 2: Comparison of ETBR expression across sex and age groups.

  • ETBR expression higher in young women (YW) than postmenopausal women (PMW) and higher in YW than YM, with aging diminishing the sex difference in ETBR expression (female vs male differences attenuate with age).

• Overall visual takeaway: In men, ETBR expression in endothelial cells is preserved with aging, while endothelial function declines; in women, ETBR expression declines with menopause and correlates positively with FMD.

Discussion and interpretation

• Main conclusion: Aging does not alter endothelial ETBR protein expression in men, contrasting with prior findings in women. This supports sex differences in the endothelin system and its regulation of vascular function.

• Mechanistic context:

  • ET-1 system becomes more active with aging in men, with greater ET-1 protein expression in endothelial cells and higher ET-1 vasoconstrictor tone largely mediated by ETAR, contributing to reduced endothelium-dependent dilation.

  • Endothelial ETBR may be more protective in women due to higher baseline ETBR prevalence or regulation, whereas men rely more on ETAR-mediated vasoconstriction; thus, age-related vascular aging may be driven more by ETAR and ET-1 in men.

• The role of ETBR in vasodilation vs vasoconstriction:

  • ETBR on endothelial cells mediates vasodilation partly via nitric oxide and prostacyclin pathways; ETBR on smooth muscle cells can mediate vasoconstriction when stimulated by ET-1.

  • The preserved ETBR expression in older men suggests that loss of ETBR is not a primary driver of aging vascular dysfunction in men, directing attention to ETAR activity and ET-1 availability.

• Testosterone and vascular aging:

  • Observed lower testosterone in OM, consistent with aging, but no robust overall correlation between testosterone and FMD in this study.

  • Exploratory analyses hint that higher testosterone within OM might associate with higher FMD, aligning with some literature that androgens modulate vascular function; however, causality and mechanisms remain to be elucidated.

  • Testosterone may influence ET-1 pathways; for example, testosterone has been shown to upregulate ET-1 in some contexts, or modify vasoconstrictor tone via ETAR; results here do not establish a direct link with ETBR expression.

• Metabolic factors and ETBR:

  • ETBR expression negatively correlated with fasting glucose, suggesting a potential metabolic-vascular interaction where glycolytic status may modulate endothelial ETBR or reflect broader endothelial health.

  • LDL and total cholesterol were higher in OM, consistent with age-related shifts in cardiometabolic risk, but ETBR expression did not significantly correlate with LDL in this cohort.

• Exercise and vascular aging:

  • Regular aerobic exercise is known to reduce ETAR vasoconstrictor tone and improve endothelial function, but this study did not detect a clear link between self-reported exercise frequency and ETBR expression or FMD, highlighting the need for more precise measures of exercise dose and type.

• Broader significance:

  • Findings emphasize sex-specific differences in ET-1 signaling with aging, with potential implications for sex-tailored prevention and treatment of age-related CVD.

  • The preserved ETBR expression in men shifts focus to ETAR-mediated pathways and ET-1 availability as contributors to endothelial aging in men.

Limitations

• Cell source: ETBR expression was measured in venous endothelial cells, not arterial endothelium. Although venous and arterial endothelial markers often correlate, arterial data would strengthen conclusions about systemic vascular aging.

• Receptor localization: ETBR is present on both endothelial and smooth muscle cells; this study assessed endothelial ETBR, but ETBR/ETAR balance across cell types could influence vascular tone and overall function.

• Sample size and power: Although robust for an invasive study, sample sizes for subgroup analyses (e.g., testosterone stratification) were limited, potentially underpowering some correlations.

• Exercise assessment: Self-reported exercise frequency lacks precision; future work should use objective metrics (e.g., VO2 max) to parse the interaction between physical activity and ETBR expression/function.

• Cross-sectional design: Causality cannot be inferred; longitudinal data would help determine whether ETBR expression changes with aging within individuals.

Conclusions

• Aging does not alter endothelial ETBR protein expression in men, contrasting with women where ETBR decreases with age/post-menopause.

• The results support sex differences in the endothelin system and suggest that ETBR regulation of vascular control is more prominent in women, whereas men may be more influenced by ETAR-driven vasoconstriction and ET-1 availability.

• Understanding which mechanisms contribute to endothelial function with aging is critical for developing strategies to prevent or offset cardiovascular disease in older adults.

NEW & NOTEWORTHY

• Prior work showed age-related declines in endothelial function in women associated with reduced ETBR in endothelial cells; this study demonstrates that ETBR expression is preserved with aging in men.

• The data add evidence for sex differences in the endothelin system and suggest endothelial ETBR regulation as a female-specific mechanism influencing vascular aging.

Connections to foundational concepts and real-world relevance

• Endothelial function as a predictor of cardiovascular risk: FMD serves as a noninvasive proxy for endothelial health; age-related declines in FMD are a known risk indicator for CVD.

• Endothelin system as a therapeutic target: ET-1 signaling contributes to vascular tone; aging and sex differences influence the balance between vasoconstriction (ETAR) and vasodilation (ETBR on endothelium).

• Implications for personalized medicine: Sex-specific differences in ET-1 signaling may inform tailored prevention strategies for aging-related vascular aging and CVD risk.

Key numerical references recap

• Participant groups:

  • YM: $n=17, ext{ age }=24 ext{ ± } 5$; BMI $24 ext{ ± } 2$; BP SBP $122 ext{ ± } 10$; DBP $70 ext{ ± } 6$; Total testosterone $9.10 ext{ ± } 2.68$ ng/mL; FMD 6.40 ext{ ± } 2.68 ext{%}; ETBR ≈ $0.42 ext{ ± } 0.17 ext{ AU}$.

  • OM: $n=14, ext{ age }=60 ext{ ± } 6$; BMI $26 ext{ ± } 3$; BP SBP $123 ext{ ± } 11$; DBP $77 ext{ ± } 7$; Total testosterone $6.28 ext{ ± } 4.21$ ng/mL; FMD 3.85 ext{ ± } 1.51 ext{%}; ETBR ≈ $0.39 ext{ ± } 0.17 ext{ AU}$.

• ETBR expression difference: $P=0.66$ (no significant difference between OM and YM).

• FMD difference: P<0.01 (OM vs YM).

• Testosterone difference: $P=0.03$ (OM lower than YM).

• Glucose difference (subset): higher in OM; specific values not provided in summary, but notable correlation with ETBR expression: $r ext{ (ETBR vs glucose)} <br>ightarrow ext{approx } -0.49; P ext{(corrected sign)} = 0.04$.

• Correlations:

  • ETBR vs FMD: trend toward inverse relation; $r
    eq 0$ with P ≈ 0.058 (reported as r ≈ -0.39).

  • ETBR vs LDL/HDL: not significantly correlated in overall interpretation.

• Figure-related sample sizes: ETBR expression measured in ~12 OM and ~15 YM; FMD analyzed with ~13 OM and ~14 YM.

References to methods and prior work (contextual)

• ETB receptor and endothelin-1 signaling in aging and sex differences have been described in foundational papers (e.g., ETAR/ETBR roles, ET-1 source, and receptor localization).

• Guidelines for FMD assessment cited in the methods (Thijssen et al., 2019).

• Prior work showing ETBR reduction in endothelial cells contributing to endothelial dysfunction in postmenopausal women (Kuczmarski et al., 2020).

Implications for exam-style understanding

• Distinguish between sex-specific mechanisms of endothelial aging: ETBR decreases in women but not in men.

• Recognize that preserved ETBR expression in men does not preclude aging-related endothelial dysfunction, implicating other pathways (notably ETAR-mediated vasoconstriction and ET-1 availability).

• Understand how normalization to HUVECs helps control staining variability in immunocytochemistry-based receptor expression studies.

• Be able to discuss how testosterone status might interact with ET-1 signaling and vascular aging, including potential mediation roles and the need for larger, more controlled studies.

The results of the experiment showed several key findings:

1. Participant Characteristics:

   • Older men (OM) were, by design, significantly older than younger men (YM).

   • OM had higher levels of total cholesterol, LDL, HDL, and fasting glucose compared to YM.

   • OM also had higher diastolic blood pressure (DBP) and lower total testosterone levels than YM.

2. Endothelial Function (FMD):

   • Flow-mediated dilation (FMD) was significantly lower in older men (3.85 ext{ ± } 1.51 ext{%}) compared to younger men (6.40 ext{ ± } 2.68 ext{%}), indicating age-related endothelial dysfunction (P < 0.01).

3. Endothelial ETBR Protein Expression:

   • Contrary to the primary hypothesis and findings in women, endothelial ETBR protein expression did not differ significantly between older men ($0.39 ext{ ± } 0.17 ext{ AU}$) and younger men ($0.42 ext{ ± } 0.17 ext{ AU}$) ($P = 0.66$).

4. Correlations:

   • There was a trend toward an inverse relationship between ETBR expression and FMD (lower ETBR associated with higher FMD), but it was not statistically significant ($r \approx -0.39; P \approx 0.058$).

   • ETBR expression showed a negative correlation with fasting glucose (higher ETBR associated with lower glucose; $r \approx -0.49; P \approx 0.04$).

   • ETBR expression was not significantly correlated with BMI, exercise frequency, cholesterol measures, or total testosterone.

In summary, while older men exhibited impaired endothelial function (reduced FMD) and several adverse cardiometabolic changes, their endothelial ETBR protein expression was preserved with aging, which contrasts with previous findings in women where ETBR expression was

The results of the experiment showed several key findings:

1. Participant Characteristics:

   • Older men (OM) were, by design, significantly older than younger men (YM).

   • OM had higher levels of total cholesterol, LDL, HDL, and fasting glucose compared to YM.

   • OM also had higher diastolic blood pressure (DBP) and lower total testosterone levels than YM.

2. Endothelial Function (FMD):

   • Flow-mediated dilation (FMD) was significantly lower in older men (3.85 ext{ ± } 1.51 ext{%}) compared to younger men (6.40 ext{ ± } 2.68 ext{%}), indicating age-related endothelial dysfunction (P < 0.01).

3. Endothelial ETBR Protein Expression:

   • Contrary to the primary hypothesis and findings in women, endothelial ETBR protein expression did not differ significantly between older men ($0.39 ext{ ± } 0.17 ext{ AU}$) and younger men ($0.42 ext{ ± } 0.17 ext{ AU}$) ($P = 0.66$).

4. Correlations:

   • There was a trend toward an inverse relationship between ETBR expression and FMD (lower ETBR associated with higher FMD), but it was not statistically significant ($r \approx -0.39; P \approx 0.058$).

   • ETBR expression showed a negative correlation with fasting glucose (higher ETBR associated with lower glucose; $r \approx -0.49; P \approx 0.04$).

   • ETBR expression was not significantly correlated with BMI, exercise frequency, cholesterol measures, or total testosterone.

In summary, while older men exhibited impaired endothelial function (reduced FMD) and several adverse cardiometabolic changes, their endothelial ETBR protein expression was preserved with aging, which contrasts with previous findings in women where ETBR expression was

The results of the experiment showed several key findings:

1. Participant Characteristics:

   • Older men (OM) were, by design, significantly older than younger men (YM).

   • OM had higher levels of total cholesterol, LDL, HDL, and fasting glucose compared to YM.

   • OM also had higher diastolic blood pressure (DBP) and lower total testosterone levels than YM.

2. Endothelial Function (FMD):

   • Flow-mediated dilation (FMD) was significantly lower in older men (3.85 ext{ ± } 1.51 ext{%}) compared to younger men (6.40 ext{ ± } 2.68 ext{%}), indicating age-related endothelial dysfunction (P < 0.01).

3. Endothelial ETBR Protein Expression:

   • Contrary to the primary hypothesis and findings in women, endothelial ETBR protein expression did not differ significantly between older men ($0.39 ext{ ± } 0.17 ext{ AU}$) and younger men ($0.42 ext{ ± } 0.17 ext{ AU}$) ($P = 0.66$).

4. Correlations:

   • There was a trend toward an inverse relationship between ETBR expression and FMD (lower ETBR associated with higher FMD), but it was not statistically significant ($r \approx -0.39; P \approx 0.058$).

   • ETBR expression showed a negative correlation with fasting glucose (higher ETBR associated with lower glucose; $r \approx -0.49; P \approx 0.04$).

   • ETBR expression was not significantly correlated with BMI, exercise frequency, cholesterol measures, or total testosterone.

In summary, while older men exhibited impaired endothelial function (reduced FMD) and several adverse cardiometabolic changes, their endothelial ETBR protein expression was preserved with aging, which contrasts with previous findings in women where ETBR expression was. not maintained as effectively. This suggests a potential gender-specific mechanism in the regulation of ETBR expression, indicating that therapeutic strategies targeting ETBR may need to be tailored differently for men and women.

User: true or false Endothelin-1 (ET-1) is generally linked to reduced flow-mediated dilation (FMD) in older men.

Model Answer: True. The background concepts section states: "prior data show higher ET-1 in older men and greater endothelial ET-1 in older vs younger men, linked to reduced flow-mediated dilation (FMD)."

User: true or false ETBR regulation may be more important for vascular health in women than in men.

Model Answer: True. The note explicitly states in the 'Discussion and interpretation' and 'Conclusions' sections that ETBR regulation of vascular control is more prominent, or a female-specific mechanism influencing vascular aging, in women compared to men.

User: true or false The study showed a strong, positive correlation between exercise frequency and ETBR protein expression in men. In other words, if exercise frequency is high, ETBR protein expression is high and vice versa.

Model Answer: False. The study explicitly states that ETBR expression was not significantly correlated with exercise frequency in men. It also mentions that exercise frequency did not show a clear association with ETBR expression or FMD in the sample.

User: truw or false Increased ETAR-mediated vasoconstriction may contribute to reduced endothelial function in aging men.

Model Answer: True. The discussion explicitly states that "ET-1 system becomes more active with aging in men, with greater ET-1 protein expression in endothelial cells and higher ET-1 vasoconstrictor tone largely mediated by ETAR, contributing to reduced endothelium-dependent dilation."