Identifying Sex-Specific Angiogenic Regulators of Impaired Adipose Tissue Expansion

Introduction:: Obesity has become a global epidemic, affecting over 650 million adults worldwide, and is associated with numerous metabolic comorbidities that pose significant public health challenges. Metabolically unhealthy obesity has been attributed to a failure in the capacity for adipose tissue expansion, which is linked to impaired adipose tissue angiogenesis [1, 2]. Therefore, identifying angiogenic regulators responsible for this impaired adipose tissue expansion is critical in developing targeted therapies for metabolically unhealthy obesity. Additionally, the shortage of sex-specific animal data for obesity is widely recognized as most studies are done with male rodents because females are resistant to obesity and metabolic disorders [3]. Therefore, we addressed the need by establishing sex-specific mouse models that are reflective of metabolically healthy obesity (MHO) vs. metabolically unhealthy obesity (MUO). We specifically investigate the protein expression of the two major angiogenic regulator families—vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGFs) and their receptors (VEGFRs and PDGFRs)—in adipose tissue and its endothelial cells (ECs).


Materials and Methods:: Male and female C57BL/6J mice were placed on either a low-fat diet (LFD, 10% fat) or a high-fat diet (HFD, 60% fat) for 12 weeks from the age of 6 weeks. After 12 weeks, five mice from each sex–diet group were euthanized to collect gonadal white adipose tissue (gWAT) for flow cytometry and ELISA assays and liver samples for metabolic profile analysis. Quantitative flow cytometry was used to measure the numbers of VEGFR-1, -2, -3, and PDGFR-α and -β molecules on the plasma membranes of ECs and macrophages [4]. ELISA was used to measure the concentrations of VEGF, placental growth factor (PlGF), and PDGF-AA, -BB, and -AB in gWAT. The remaining LFD mice continued an LFD for another 12 weeks. The HFD mice were split into two groups: Group 1 continued an HFD for more weight gain, and Group 2 switched to LFD for weight loss. At the end of week 24, the continued HFD male group showed significant fatty liver, which is a hallmark of metabolically unhealthy obesity, as previously reported [5,6]. The mice were euthanized for quantitative flow cytometry and ELISA studies.


Results, Conclusions, and Discussions:: We established mouse models reflective of MHO and MUO phenotypes, with 24-week HFD males showing signs of MUO, including gWAT reduction (Fig. A) and fatty livers, as previously reported [5,6]. Meanwhile, 24-week HFD females and 12-week HFD males and females did not show the MUO signs and were considered MHO. These outcomes align with previous research showing that male rodents develop MUO after 12 weeks of HFD [5], while females are more resistant [3]. We also included weight-loss models, which increased gWAT weight by ~50% after a 12-week HFD and restored their LFD gWAT phenotype with an HFD-to-LFD change from Week 13-24 (Fig. A). We measured a significantly lower number of VEGFR1 on the plasma membrane surface of adipose ECs (2000 VEGFR1s/cell) in LFD mice and HFD MUO mice, compared with 3,000–5,000 VEGFR1s/cell in HFD MHO mice (Fig. B). This significant upregulation is not seen with the other VEGFRs and PDGFRs. Thus, the membrane VEGFR1 upregulation may promote gWAT expansion, preventing the transition to MUO phenotype. Females displayed significant downregulation (p < 0.05) of PlGF (0.1 to 0.02 ng/mg protein), PDGF-AA (0.1 to 0.02 ng/mg), and PDGF-AB (7 to 3 ng/mg) during gWAT expansion, compared with the controls, while males maintained consistent levels of these ligands in gWATs. Previous studies show that serum PlGF level was higher in MUO than non-obese individuals (0.0141 vs. 0.0109 ng/mL, p < 0.05) [7], while serum PDGF-AA level was lower in MUO than non-obese individuals (18.3 vs. 23.5 ng/mL, p < 0.01) [8]. However, these studies did not consider sex-specific differences.

Our data suggest that VEGFR1 upregulation is a potential angiogenic treatment for restoring MHO phenotype, while females may need additional inhibitors to downregulate PlGF, PDGF-AA, and PDGF-AB in gWAT. Through our mouse models, we revealed the sex-specific angiogenic regulations responsible for gWAT expansion, bringing us closer to having an angiogenic target for restoring MHO status. Our data also encourages systems biology computational models to delineate the obesity-angiogenesis interface, advancing angiogenic therapy for treating MUO. Liver metabolic profiling and snRNAseq studies are underway to elucidate the connections between sex-specific angiogenic and metabolic dysregulations.


Acknowledgements (Optional): : The research reported in this publication was supported by the National Institute of Heart, Lung, and Blood of the National Institutes of Health under Award Number 7R01HL159946-02. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


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