Strain- and sex-dependent variability in hepatic microcirculation and liver function in mice

Abstract

BACKGROUND

The integrity and functionality of the hepatic microcirculation are essential for maintaining liver health, which is influenced by sex and genetic background. Understanding these variations is crucial for addressing disparities in liver disease outcomes.

AIM

To investigate the sexual dimorphism and genetic heterogeneity of liver microcirculatory function in mice.

METHODS

We assessed hepatic microhemodynamics in BALB/c, C57BL/6J, and KM mouse strains using laser Doppler flowmetry and wavelet analysis. We analyzed the serum levels of alanine transaminase, glutamic acid aminotransferase, total bile acid, total protein, alkaline phosphatase, and glucose. Histological and immunohistochemical staining were employed to quantify microvascular density and the expression levels of cluster of differentiation (CD) 31, and estrogen receptor α, and β. Statistical analyses, including the Mantel test and Pearson correlation, were conducted to determine the relationships among hepatic function, microcirculation, and marcocirculation between different sexes and across genetic backgrounds.

RESULTS

We identified sex-based disparities in hepatic microhemodynamics across all strains, with males exhibiting higher microvascular perfusion and erythrocyte concentration, but lower blood velocity. Strain-specific differences were evident, particularly in the endothelial oscillatory characteristics of the erythrocyte concentration. No sex-dependent differences in estrogen receptor expression were observed, while significant variations in CD31 expression and microvascular density were observed. The correlations highlighted relationships between hepatic microhemodynamics and liver function indicators.

CONCLUSION

Our findings indicate the influence of genetic and sex differences on hepatic microcirculation and liver function, highlighting the necessity of incorporating both genetic background and sex into hepatic physiology studies and potential liver disease management strategies.

Key Words: Hepatic microhemodynamics; Sex differences; Mouse strains; Biological oscillators; Hepatic microcirculation

Core Tip: This study reveals significant strain and sex-dependent variations in hepatic microcirculation among murine, highlighting the implications for liver health. Male mice exhibited higher microvascular perfusion and erythrocyte concentration, while sex-specific differences in endothelial function were indicated across strains. Cluster of differentiation 31 expression linked to microvascular density varied by sex, suggesting a role in hepatic microhemodynamics. These findings suggest the necessity of integrating genetic and sex factors into the understanding of liver physiology and pathology, potentially guiding personalized therapeutic strategies.

INTRODUCTION

The liver, as the largest visceral organ in humans, accounts for approximately 2.5 % of the total body weight and receives approximately 25 % of cardiac output. This robust blood supply is predominantly sourced from two major vessels, with the portal vein contributing 70%-80% of the inflow by delivering nutrient-rich blood, while the hepatic arteries supply the remaining 20%-30% of oxygenated blood. These vessels undergo extensive bifurcation, culminating in terminal hepatic arterioles and portal venules, with diameters ranging from 15 μm-35 μm and lengths of 50 μm-70 μm[1]. This network ultimately feeds into the hepatic sinusoids, the critical components of the liver microvasculature. Sinusoids are characterized by a specialized fenestrated endothelium that not only increase permeability but also facilitates dynamic interactions between blood and hepatocytes[2], thereby playing a vital role in liver function and homeostasis.

The essential functions of the liver in biosynthesis, metabolism, detoxification, and immune surveillance are fundamentally reliant on the integrity of its microcirculation. This vascular system ensures the effective delivery of nutrients and oxygen to parenchymal tissues and plays a vital role in regulating vascular tone, facilitating leukocyte trafficking during hepatic inflammation, and enabling the clearance of toxins and pathogens from the bloodstream. Emerging evidence indicates that biological sex is a modifying factor of hepatic immune regulation, thereby contributing to hepatic immune outcomes in health and disease[3]. Additionally, genetic variations may alter the composition of liver-associated microbial DNA, further complicating the communication between host and microbial factors in liver function[4]. These lines of evidence represent potentially actionable mechanisms of disease biology. Disruptions in hepatic microvascular circulation emerge as critical factors in liver pathology, leading to organ injury or failure in ischemic and inflammatory diseases[5,6]. These disruptions are also associated with elevated mortality rates among affected individuals.

Moreover, the manifestation and progression of liver disease are significantly influenced by sex-based and genetic disparities. Research, including studies by Ruhl et al[7], has shown that men exhibit a higher prevalence of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) than women[8,9]. Women with NAFLD typically present a less atherogenic lipid profile and a more favorable cardiovascular risk[10]. Additionally, males are more susceptible to hepatic fibrosis, which correlates with an increased risk of inflammation-driven HCC[11,12]. Ethnic and racial differences further complicate the epidemiology of liver disease; for instance, the prevalence of NAFLD is highest in Europe (28.04%), followed by East Asia (19.24%) and the Middle East (12.95%)[13]. Genetic factors, particularly variants in the Patatin-like phospholipase domain-containing protein 3 (PNPLA3), add another layer of complexity, with varying frequencies of the rs738409 GG genotype observed across different populations[14].

Understanding the relationship between hepatic microhemodynamics and liver function is essential for unraveling the complex interplay of these physiological and pathological processes. Variations in microvascular structure and function, influenced by genetic and sex-based differences, can significantly impact hepatic perfusion, nutrient delivery, and metabolic regulation. These physiological disparities are crucial for maintaining liver homeostasis and may elucidate disparities in disease susceptibility and progression. The aim of our study was to investigate the relationship between hepatic microhemodynamics and liver function, with a particular focus on variations attributable to sex and genetic background.

MATERIALS AND METHODS

Animals

This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Microcirculation, Chinese Academy of Medical Sciences (CAMS) (Approval No. CAMS-IM-IACUC-2022-AE-0917). C57BL/6J mice are foundational in liver studies due to their well-defined genetics and reliable responses to models[15]. Details on liver fibrosis and inflammation can be obtained by studying BALB/c mice, which exhibit distinct metabolomic and immunological profiles[16]. KM mice, though less common, are valuable for research on oxidative stress and hepatotoxicity[17]. These strains facilitate the investigation of hepatic microhemodynamics across diverse genetic backgrounds. All mice aged 8 weeks (n = 6 each group), acquired from the Institute of Laboratory Animal Science, CAMS, Beijing, China, were housed under controlled conditions (temperature: 18-22 °C, humidity: 50%-65%, 12-hour/12-hour light/dark cycle) with ad libitum access to food and water. We employed a randomized block design to allocate the mice to experimental groups, controlling for baseline variations and ensuring balanced, comparable groups.

Monitoring of blood pressure

Blood pressure measurements were performed via a noninvasive tail-cuff method with the Intelligent Sphygmomanometer BP-2010A (Softron Biotechnology, Beijing, China). This method is established as equivalent to invasive techniques, showing strong correlations with intra-arterial measurements[18]. We chose this approach to avoid potential confounding effects on microhemodynamics associated with invasive methods. The mice were acclimatized for 5 minutes in a restraint device placed on a heated pad (37 °C) before systolic, diastolic, and mean arterial pressures were recorded over three consecutive trials. Blood glucose levels were determined via tail snip using the One Touch Ultra glucometer (LifeScan, Johnson and Johnson, Milpitas, CA, United States).

Determination of the integrated hepatic microcirculatory profile

To investigate hepatic microhemodynamics, we employed a laser Doppler blood perfusion monitoring system (Virtual machines, Moor Instruments, Ltd., Axminster, United Kingdom), which was calibrated with standard microspheres prior to the experiments. Following acclimatization for 10 minutes, the mice were initially anesthetized via inhalation of 2% isoflurane (R510-22; RWD Life Science Co., Shenzhen, China) in a 50% oxygen mixture through a small animal anesthesia machine (matrix VMR; Midmark Corporation, OH, United States). An incision was precisely made along the medioventral line to gently expose the entire liver, and the Versa-probe 4 (Moor Instruments) was stably positioned on the exposed hepatic tissue with the assistance of a probe holder (Moor Instruments). Three sites were selected, and measured for 1 minute each, and the backscattered light collected by the probe was processed by analog and digital signals to generate hepatic microhemodynamics. The microcirculatory blood distribution pattern was revealed in the scatter plot. The microvascular hemorheological parameters including microvascular blood flux, microvascular erythrocyte concentration, and microvascular blood velocity were analyzed quantitatively as functional evaluation of hepatic microhemodynamics.

Wavelet transform analysis

Wavelet transform analysis is superior to traditional Fourier analysis for blood flow signals due to its ability to handle non-stationary signals and provide detailed time frequency information. The microhemodynamic signals were subjected to oscillations ranging from 0.005 Hz-5.0 Hz, including nitric oxide (NO)-independent endothelial signals (0.005 Hz-0.0095 Hz), reflecting baseline endothelial function; NO-dependent endothelial signals (0.0095 Hz-0.04 Hz), representing responses regulated by NO; neurogenic signals (0.04 Hz-0.15 Hz), linked to neural influences on microhemodynamics; myogenic signals (0.15 Hz-0.4 Hz), representing smooth muscle responses to blood pressure changes; respiratory signals (0.4 Hz-2.0 Hz), correlating with breathing patterns; and cardiac signals (2.0 Hz-5.0 Hz), which are associated with heart rate and rhythm[19,20]. Each of these signals is related to a specific physiological influence modulating the hepatic microcirculation response. Wavelet transformation was performed to create the representation of signals measured from hepatic microhemodynamics in the time frequency domain and estimate the contribution of rhythmical components in blood flow signal. Using the Morlet wavelet, a Gaussian window was scaled and shifted over time, while amplitudes were computed on the basis of average wavelet coefficients, thus the spectral attributes of the oscillators were derived. A spectral scalogram was then generated. Variables such as time (second), frequency (Hz), and spectral amplitude (AU) were situated within the coordinate system. Finally, the amplitudes of the six oscillators were compared across the different groups.

Liver function evaluation

Blood samples were collected from the inferior vena cava and then centrifuged at 3000 rpm for 20 minutes to obtain the serum. Subsequent biochemical analyses were conducted at the clinical laboratory of Peking Union Medical College Hospital, utilizing an automated biochemical analyzer (Beckman Coulter, United States) and an automatic protein analyzer (Siemens, Germany). The serum levels of alanine aminotransferase (ALT) (measured with the lactate dehydrogenase method), aspartate aminotransferase (AST) (measured with the malate dehydrogenase method), alkaline phosphatase (ALP) (measured with the nitrophenyl phosphate substrate-adenosine monophosphate buffer method), glucose (Glu) (measured with the hexokinase method), total protein (TP) (measured via biuret colorimetry) and total bile acid (TBA) (measured with an enzymatic cycling assay) were quantitatively determined. The selected markers were chosen because of their ability to reflect liver health and dysfunction. Each marker serves a distinct role in liver function, and tests to measure the levels of these markers are crucial for diagnosing liver diseases and monitoring their progression. ALT and AST are key indicators of hepatocellular injury, with elevated levels indicating liver cell damage. Elevated ALP levels indicate the potential for cholestasis or bile duct obstruction, while abnormal Glu levels indicate impaired metabolic function. TP levels, particularly albumin levels, are indicative of the synthetic capacity of the liver, with decreased levels indicating chronic disease. TBA levels reflect the ability of the liver to process bile acids; elevated levels suggest dysfunction. Reference intervals for the evaluated hepatic function parameters in mice were established on the basis of previous literature[21-23].

Histology and immunohistochemistry

For morphological studies, tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick slices using a microtome (Leica Biosystems, Germany) for hematoxylin and eosin and immunohistochemical staining. For antigen retrieval, the slices were deparaffinized, rehydrated, and incubated in boiling 10 mmol/L citrate buffer (potential of hydrogen = 6.0; Zhongshan Golden Bridge Biotechnology, Beijing, China). The sections were subsequently incubated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity, followed by blocking with 3% bovine serum albumin (TBD Science Technology, Tianjin, China) in phosphate-buffered saline. The sections were incubated in blocking buffer overnight at 4 °C, after incubating with primary mouse monoclonal antibodies against cluster of differentiation (CD) 31 (diluted 1:100; Santa Cruz Biotechnology), mouse estrogen receptor α (ERα) and estrogen receptor β (ERβ) monoclonal antibodies (diluted 1:25; Abcam). Horseradish peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) and 3,3’-diaminobenzidine tetrahydrochloride solution (Zhongshan Golden Bridge Biotechnology, Beijing, China) were subsequently used for the incubation of sections, after which the section were dehydrated and mounted. An antibody mixture (Ambiping Medical Technology, Guangzhou, Guangdong Province, China) was used as an isotype control. Positive staining was detected using a Leica DFC450 microscope (Leica Microsystems, Leitz, Germany). For quantitative evaluation, ImageJ software (National Institutes of Health, Bethesda, MD, United States) was used to capture and analyze the digital images of the stained sections. In addition, the intensity of CD31, ERα and ERβ positive staining was quantified by calculating the integrated density. Microvascular density (MVD) was determined and quantified through manual counting of six random × 40 fields to obtain an average. In selecting these fields, we ensured optimal staining with vascular markers (CD31, ERα, and ERβ) for effective visualization, encompassing diverse tissue regions while excluding artifacts and avoiding overlapping fields.

Statistical analysis

All the measurements were performed in duplicate to ensure reliability. The statistical analysis was conducted with GraphPad Prism software (version 8.02; GraphPad Software, CA, United States). The data derived from laser Doppler signals and wavelet analysis were presented as the mean ± SEM. We employed two-factor analysis of variance (ANOVA) with interaction effects, incorporating sex and strain as independent variables. In cases of non-significant overall ANOVA results, we refrained from post-hoc comparisons to avoid misleading conclusions, and conducted further analyses only when significant main effects or interactions were observed, with Tukey’s method applied for multiple comparisons to control the familywise error rate. Statistical significance was assessed using a predefined alpha threshold of 0.05. To elucidate the relationships between hepatic function and hepatic microhemodynamics in three murine strains, segregated by sex, both the Mantel test and Pearson correlation analysis were utilized. The Mantel test was used to assess relationships between distance matrices, especially those relevant to genetic divergence, while Pearson correlation was used to linear relationships. Pearson’s correlation coefficients (r) were considered relevant for Pearson’s P < 0.05 and values of r > 0.4 or r < -0.4.

RESULTS

Sexual dimorphism in hepatic microvascular perfusion across murine strains

Sex-specific differences in hepatic microvascular blood flux were evident, with males consistently demonstrating higher perfusion levels across all strains (all P < 0.001), suggesting a sex-determined influence on the variability of hepatic microvascular blood flux. Conversely, no significant sex differences in respiratory oscillations in hepatic microvascular blood flux were observed within each strain (Supplementary Table 1), although female KM mice exhibited greater oscillatory activity than their BALB/c and C57BL/6J counterparts did (P < 0.01) (Figure 1), suggesting strain-dependent modulation of hepatic microcirculation. Collectively, these findings illuminate the substantial heterogeneity in hepatic microvascular perfusion that may affect the responsiveness of the liver to physiological demands.

Figure 1 Comparative analysis of hepatic microcirculatory blood perfusion and characteristic oscillatory amplitudes between sexes and across three mouse strains. A: Hepatic microcirculatory blood perfusion patterns in the BALB/c, C57BL/6J, and KM mouse strains. The rectangular insert highlights the extracted pattern of microcirculatory blood distribution; B and C: Two-dimensional and three-dimensional spectral scalograms representing the characteristic amplitudes of hepatic microcirculation profiles; D: Quantitative analysis of characteristic oscillatory amplitudes in hepatic microcirculatory blood perfusion profiles. The data are presented as the mean ± SEM. n = 6 each group. ^aP < 0.05. ^cP < 0.001. AU: Amplitude; NO: Nitric oxide; NS: Not significant; PU: Perfusion unit.

Disparities in hepatic microvascular erythrocyte concentration by sex and strain

Erythrocytes regulate endothelial and microvascular functions through the modulation of oxygen delivery. Significant sexual dimorphism of the erythrocyte concentration within the hepatic microvasculature was observed across all strains (all P < 0.001) (Supplementary Table 2). Compared with females, males of the C57BL/6J and KM strains presented increased erythrocyte concentrations, however, in BALB/c mice, where this trend was reversed (Figure 2A). Detailed analysis of endothelial oscillator amplitudes revealed that, compared with KM females, KM males had lower amplitudes in the endothelial frequency range (0.01 Hz-0.04 Hz), whereas C57BL/6J females presented the highest amplitudes (Figure 2B). KM males presented the lowest amplitudes relative to those of other strains (Figure 2C). Quantitative analysis of sex-related disparities in erythrocyte concentration oscillators at corresponding frequencies (Figure 2D) revealed that KM males presented increased enhanced endothelial oscillatory activity. Specifically, significant differences in both NO-independent (P < 0.05) and NO-dependent (P < 0.05) endothelial amplitudes were detected between KM females and males. Although no significant differences were revealed across other variables within the BALB/c and C57BL/6J strains, significant differences were identified in NO-independent endothelial oscillator amplitudes between C57BL/6J and KM males (P < 0.05). Collectively, these findings indicate heterogeneity in erythrocyte concentration, suggesting the multifaceted regulation of hepatic microcirculation by both sex and genetic determinants within these murine models.

Figure 2 Comparative analysis of hepatic microcirculatory erythrocyte concentration between sexes and across three mouse strains. A: Microcirculatory erythrocyte concentration in BALB/c, C57BL/6J, and KM mice. The distribution pattern of erythrocyte concentration is presented in the rectangular insert; B and C: Two-dimensional and three-dimensional spectral scalograms illustrating the characteristic amplitude of the hepatic microcirculatory erythrocyte concentration; D: Quantitative assessment of the characteristic oscillatory amplitudes from the hepatic microcirculatory erythrocyte concentration profiles. The data are presented as the mean ± SEM. n = 6 each group. ^aP < 0.05. ^cP < 0.001. AU: Amplitude; NO: Nitric oxide; NS: Not significant; PU: Perfusion unit.

Variation in hepatic microvascular blood velocity across mouse strains and between the sexes

The distribution of blood flow and erythrocyte fluxes within the hepatic microvascular network is critical for velocity. As shown in Figure 3A and Supplementary Table 3, significant variations in hepatic microvascular blood velocity were observed between the sexes and across the mouse strains. Compared with their male counterparts, female BALB/c mice presented higher microvascular velocities. Conversely, the C57BL/6J and KM strains demonstrated the opposite pattern, with males displaying lower velocities. Wavelet transform analysis revealed significant differences primarily in the amplitudes of cardiac and myogenic oscillators (Figure 3B and C). Compared with male C57BL/6J mice, female C57BL/6J mice presented greater variance in cardiac oscillator amplitudes. Figure 3D highlights the distinctive patterns in the myogenic oscillator of blood velocity, particularly in C57BL/6J and KM male mice. These findings suggest the role of sex and genetic strain in interpreting hepatic blood velocity, suggesting that both factors significantly influence associated physiological functions.

Figure 3 Comparative analysis of hepatic microcirculatory blood velocity between sexes and across three mouse strains. A: Hepatic microcirculatory blood velocity in BALB/c, C57BL/6J, and KM mice. The rectangular insert provides a detailed view of the hepatic microcirculatory blood velocity distribution pattern; B and C: Two-dimensional and three-dimensional spectral scalograms demonstrating the characteristic amplitudes of the hepatic microcirculatory blood velocity profiles; D: Quantitative analysis of the characteristic oscillatory amplitudes in the hepatic microcirculatory blood velocity profiles. The data are presented as the mean ± SEM. n = 6 each group. ^aP < 0.05. ^cP < 0.001. AU: Amplitude; NO: Nitric oxide; NS: Not significant; PU: Perfusion unit.

Histopathological analysis of sex-specific variations in the hepatic microvasculature

We subsequently performed hematoxylin and eosin staining to assess structural differences in the hepatic microvasculature. Additionally, we employed immunohistochemistry to evaluate the expression patterns of the endothelial cell marker CD31, ERα and ERβ, aiming to elucidate their potential roles in modulating these microhemodynamic variations. As shown in Figure 4A and B, Supplementary Figure 1 and Supplementary Table 4, contrary to expectations, no significant sex-based differences in the expression levels of ERα and ERβ were observed within the hepatic microvasculature. However, among the male mice, C57BL/6J mice presented significantly elevated expression levels of both ERα and ERβ compared with BALB/c and KM mice (all P < 0.05). In contrast, analysis of CD31 expression levels revealed significant sex differences across all strains. Compared with their male counterparts, female BALB/c and KM mice presented significantly greater CD31 expression levels, whereas C57BL/6J females presented a lower CD31 expression level (P < 0.01 for all comparisons). Additionally, MVD analysis indicated that males across all three strains presented greater MVD than females did (all P < 0.05, Figure 4C). Collectively, these findings suggest that CD31 plays a role in mediating sex differences in the observed microhemodynamic variations.

Figure 4 Hematoxylin and eosin staining and immunolabeling analysis of liver tissue from different sexes across BALB/c, C57BL/6J, and KM mice. A: Hematoxylin and eosin (HE) and immunohistochemical staining of estrogen receptor α (ERα), and estrogen receptor β (ERβ), and cluster of differentiation (CD) 31 in the liver. The scale bar represents 50 μm. The insert in the lower panel provides a detailed representation of liver tissues at a higher magnification. Scale bar = 10 μm; B: HE staining of liver tissue sections revealing morphological details. Scale bar = 200 μm. The insert in the lower panel provides a detailed representation of liver tissues at a higher magnification. Scale bar = 50 μm; C: Quantitative analysis of the protein expression levels of ERα, ERβ, and CD31 and the microvascular density in the livers of BALB/c, C57BL/6J, and KM mice. For each group, n = 6 samples, and six microscopic fields of view were selected for each sample. The data are presented as the mean ± SEM. n = 6 each group. ^aP < 0.05. ^bP < 0.01. NS: Not significant; Erα: Estrogen receptor α; Erβ: Estrogen receptor β; CD31: Cluster of differentiation 31.

Correlation analysis of liver function indictors and hepatic microhemodynamics

To exclude the confounding effects of body weight heterogeneity on microhemodynamics, we employed a dual analytical approach comprising two-way ANOVA and correlation analysis. Two-way ANOVA revealed significant differences and interactions in body weight [F-ratio (2, 30) = 5.427; P = 0.009] (Supplementary Tables 5 and 6). Male C57BL/6 mice presented significantly greater body weights than females did (P < 0.0001), a trend not observed for the BALB/c and KM strains. KM mice consistently surpassed both C57BL/6 and BALB/c mice in terms of body mass, irrespective of sex (all P < 0.0001). These findings highlight the influence of genetic strain and sex on phenotypic diversity, however, these weight differences do not directly correlate with changes in hepatic microhemodynamics (Figure 5).

Figure 5 Relationship among hepatic microhemodynamics, liver function and macrohemodynamics. A: Cross-comparison of liver function between different mouse strains and sexes. The established reference values for each of the five indicators are marked with dashed lines, with triangles serving as indicating markers. Yellow represents females, whereas blue signifies males; B: Interrelationships among the mean amplitudes of the three microhemodynamic indicators and macrohemodynamic indicators were analyzed using Pearson correlation analysis. The correlations among liver function indicators, hepatic microhemodynamics, and blood pressure and heart rate were shown respectively with connecting lines indicating these relationships. Microhemodynamics indicators: Flux, concentration, and speed; Liver function indicators: Alanine aminotransferase, aspartate aminotransferase, total protein, alkaline phosphatase, total bile acid, and glucose; Macrohemodynamics indicators: Heart rate, systolic blood pressure, mean arterial pressure, and diastolic blood pressure. P values are represented by the color, and r values are represented by the line thickness; C: The relationships between microhemodynamics and liver function, across different mouse strains and between sexes, were independently analyzed using the Mantel test (n = 6 each group). The edge width signifies the correlation strength, while the edge color indicates statistical significance. “Micro” indicates the mean amplitude of the five characteristic frequencies of flux, concentration, and speed. The data are presented as the mean ± SEM. n = 6 each group. ^aP < 0.05. ^cP < 0.001. ^dP < 0.0001. RI: Respiratory index; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; TP: Total protein; ALP: Alkaline phosphatase; TBA: Total bile acid; Glu: Glucose; SBP: Systolic blood pressure; HR: Heart rate; DBP: Diastolic blood pressure; MBP: Mean arterial pressure.

Liver function revealed sex differences in the serum ALP and TBA levels across the three mouse strains, as shown in Figure 5A and Supplementary Table 7. Females consistently presented higher TBA levels than males did, regardless of strain. Conversely, among KM mice, females presented significantly lower serum TP levels than males did. Strain-specific differences were also identified: BALB/c females had significantly lower serum AST, TBA, and ALP levels than C57BL/6J females did (all P < 0.0001). Similarly, compared with C57BL/6J females, KM females presented lower serum TBA and ALP levels (all P < 0.0001). To investigate the potential influence of microhemodynamics on liver function, we analyzed the spectral components of microhemodynamics and their associations with hepatic parameters across sexes and strains. The significant correlations between the physiological amplitudes of hepatic microcirculation and liver function parameters are shown in Figure 5B and C and Supplementary Table 8. The ALT level was significantly positively correlated with the neurogenic amplitude of blood speed (r = 0.342, P = 0.041), and the AST level was similarly positively correlated with myogenic indicators of blood speed (r = 0.333, P = 0.047). Additionally, the TBA level was positively correlated with both the neurogenic amplitude of the erythrocyte concentration (r = 0.384, P = 0.021) and the blood speed (r = 0.400, P = 0.016). Conversely, the Glu level was negatively correlated with the respiratory amplitude of the erythrocyte concentration (r = -0.342, P = 0.041). Furthermore, similar correlations were observed between macro-circulatory microhemodynamics and liver function (Supplementary Table 9), with ALT and AST levels being correlated negatively with systolic blood pressure (r = -0.428, P = 0.009; r = -0.464, P = 0.004) and positively with diastolic blood pressure (r = 0.449, P = 0.006; r = 0.491, P = 0.006). These results suggest that the complex interplay between liver function and hepatic microhemodynamics is influenced by both sex and strain.

DISCUSSION

The liver has a complex microvascular network that is crucial for its diverse functions. The sex-based disparities in hepatic microcirculation indicate fundamental physiological adaptations and pathological vulnerabilities. Physiologically, increased microvascular perfusion and erythrocyte concentrations in male mice reflect an adaptive mechanism to meet heightened metabolic and anabolic demands[24], potentially mediated by androgen-driven modulation of endothelial function and erythropoiesis. This perfusion ensures efficient nutrient and oxygen delivery, supporting hepatocellular activity and resilience. In contrast, female mice exhibit lower perfusion rates coupled with higher blood velocity, suggesting a streamlined and efficient microvascular network possibly influenced by the vasodilatory and anti-inflammatory effects of estrogen[25,26]. Such configurations confer metabolic efficiency and increased clearance capacities, aligning with observed protective effects against metabolic liver diseases in females[27]. Pathologically, these sex-specific microcirculatory profiles have implications for liver disease susceptibility and progression. Elevated perfusion and erythrocyte load in males predispose them to increased shear stress and oxidative damage within the hepatic sinusoids, fostering an environment conducive to inflammatory cascades, fibrosis, and steatohepatitis[28-30]. The crosstalk between higher erythrocyte concentrations and reactive oxygen species (ROS) generation exacerbates hepatocellular injury, accelerating the transition from benign hepatic steatosis to severe fibrotic states. Conversely, the regulated blood flow and increased endothelial integrity in females mitigate excessive inflammatory responses and oxidative stress[31], thereby conferring resilience against progressive liver fibrosis and cirrhosis.

Our study also revealed strain-specific differences in microhemodynamics. These genetic determinants of hepatic microhemodynamics are rooted in divergent expression profiles of key regulatory proteins, including integrins, adhesion molecules[32], and intracellular signaling cascades that govern endothelial cell behavior and microvascular integrity. For example, polymorphisms in genes encoding for CD31 or estrogen receptors modulate endothelial function and responsiveness to hormonal cues, thereby influencing microvascular perfusion and erythrocyte dynamics. Additionally, strain-specific variations in mitochondrial efficiency and ROS handling could impact endothelial health and microcirculatory resilience, further diversifying hepatic responses to pathological stressors. Furthermore, genetic differences substantially influence microvascular structure and the expression of endothelial receptors and signaling pathways[33]. These findings emphasize the role of genetic determinants in shaping microvascular architecture and function, which in turn affect hepatic health. The variability in how different strains respond to similar physiological or pathological stimuli can be attributed to these genetic factors, which also correlate with the varying prevalence of hepatic conditions such as NAFLD and HCC in these models[34]. The differential expression of CD31 between sexes and across strains observed in our study indicates its role in maintaining endothelial integrity and regulating hepatic microcirculation. CD31 is essential for endothelial barrier function and leukocyte transmigration, while vascular endothelial growth factor receptor 1 (VEGFR1) orchestrates angiogenesis and macrophage recruitment during liver repair. The engagement of CD31 at sites of active vascular endothelial cell stimulation is essential for the maintenance of flow-driven physiological adjustments[35]. CD31 and VEGFR1 synergistically increase endothelial repair by modulating each other’s signaling pathways. CD31 stabilizes VEGFR1 on the endothelial surface, thereby potentiating VEGFR1-mediated angiogenic responses essential for revascularization and tissue regeneration. Additionally, CD31-induced metabolic reprogramming toward glycolysis supports the energetic demands of VEGFR1-driven angiogenesis[36], particularly under the hypoxic conditions common in hepatic injury. Furthermore, the regulation of leukocyte transmigration by CD31 influences the local production of proangiogenic factors, creating a microenvironment that facilitates VEGFR1-mediated microvascular remodeling. Sex-specific hormonal differences modulate this processing, accounting for the observed disparities in microhemodynamics.

The sex-based disparities observed in hepatic microhemodynamics, especially the elevated microvascular perfusion and density in male mice alongside higher TBA levels in females, indicate differential susceptibility to liver diseases between the sexes. Elevated microvascular perfusion and increased MVD in males may facilitate a more extensive distribution of profibrotic and oncogenic factors within the liver, potentially exacerbating conditions such as hepatic fibrosis and HCC, which are more prevalent in males clinically. This increased perfusion could be driven by testosterone-mediated upregulation of angiogenic factors such as VEGF, promoting endothelial proliferation and microvascular expansion. Conversely, higher TBA levels in females reflect efficient bile acid metabolism and clearance, supporting protective effects against lipid accumulation and insulin resistance, thereby reducing the risk of NAFLD and its progression to severe liver pathologies. Additionally, the vasoprotective and anti-inflammatory properties of estrogen may contribute to a regulated microvascular environment in females, mitigating excessive vascular remodeling and fibrosis. The lack of significant differences in estrogen receptor expression suggests that downstream signaling pathways, rather than receptor abundance, mediate these protective effects. Furthermore, the correlation between microhemodynamic data and levels of liver function markers such as ALT and AST suggest that microvascular dysfunction could serve as an early biomarker for hepatic injury[37], preceding overt biochemical disturbances[38]. This evidence provides support for sex- and genetic-specific diagnostic and therapeutic strategies, where modulating microcirculatory parameters could ameliorate disease trajectories. For example, increasing endothelial function and optimizing erythrocyte dynamics in males provide protective benefits, while leveraging estrogenic pathways in females could sustain vascular health and functional integrity. While the integration of microhemodynamic data with traditional biochemical markers holds promise for improving liver disease management[39], challenges remain. These findings suggests that microcirculatory assessments could significantly improve the early detection and monitoring of liver diseases[40], such as NAFLD and hepatic fibrosis. By integrating microhemodynamic data with traditional biochemical markers, clinicians could increase their accuracy of disease staging and progression tracking, potentially allowing timely implementation of interventions before substantial biochemical abnormalities arise.

Integrating our hepatic microhemodynamic findings with the findings of previous parallel studies on renal and intestinal microcirculation provides an overview of the systemic orchestration of microvascular function modulated by sex and genetic background[41,42]. Across the liver, kidneys, and intestine, male mice consistently exhibited elevated microvascular perfusion and erythrocyte concentrations but demonstrated lower blood velocities than females did, a phenomenon likely driven by sex hormones that influence endothelial responsiveness and vascular tone. Strain-specific differences, particularly in endothelial oscillatory characteristics, highlight the genetic predispositions that shape microvascular architecture and functionality. These organ-centric disparities reflect the interconnected nature of the systemic microcirculation, where hepatic, renal, and intestinal vessels collaboratively sustain overall physiological homeostasis, which informs the development of targeted therapeutic strategies that account for genetic and sex-related variability, thereby increasing the efficacy of interventions aimed at preserving or restoring microvascular function across multiple organ systems.

Building on our findings of sex- and strain-specific hepatic microcirculatory variations, targeted therapeutic strategies can be developed to modulate microvascular flow and improve endothelial function[43], particularly for conditions such as hepatic fibrosis and HCC[44,45]. Genetic and sex-specific profiles should be integrated in personalized medicine approaches to optimize treatments. For instance, utilizing vasodilators or NO promoting agents tailored to individual microcirculatory patterns could normalize blood flow and mitigate fibrosis progression[46]. Increasing endothelial integrity through antioxidants could prevent endothelial dysfunction and inhibit tumor angiogenesis in HCC. Additionally, genetic screening for variants such as PNPLA3 could inform the selection of microcirculatory modulators[47], ensuring therapies are aligned with genetic predispositions. Incorporating hormone-based treatments, such as selective estrogen receptor modulators for females, could further refine efficacy and reduce adverse effects. Advanced drug delivery systems, such as nanoparticle carriers targeting endothelial cells[48], promise precise intervention within the hepatic microenvironment[49], maximizing therapeutic impact while minimizing systemic toxicity.

Strengths and limitations

Our study of hepatic microcirculation across different murine strains and between sexes has several limitations. Environmental factors such as stress, microbial exposure, and diet were not fully controlled, potentially confounding our observations of strain- and sex-related differences. The mechanisms underlying the sex- and strain-based differences observed remain unknown, particularly the roles of sex hormones. Additionally, the genetic diversity represented by the three murine strains does not capture the full spectrum. Moreover, our cross-sectional approach does not account for changes over time, necessitating longitudinal studies to fully understand the dynamics of hepatic microcirculation.

CONCLUSION

Our study highlights the heterogeneous nature of hepatic microhemodynamics, which is influenced by sex and genetic background. These findings improve our understanding of the contributions of the microcirculation to hepatic health and disease and highlight the importance of incorporating sex and genetic factors into future hepatic investigations, which could lead to personalized therapeutic strategies.