Changes in Proteome Profiles Linked to Hormonal Contraceptive Use Among African American Women with untreated high blood pressure

Proteomic Alterations Associated with Oral Contraceptive Use in Hypertensive Premenopausal African American Women | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Proteomic Alterations Associated with Oral Contraceptive Use in Hypertensive Premenopausal African American Women Lisa DeRoo , Malak Abbas , Gabriel Goodney , Merry L. Lindsey , Ashton F. Oliver , Han Le , Antwi-Boasiako Oteng , View ORCID Profile Amadou Gaye doi: https://doi.org/10.1101/2024.08.12.607634 Lisa DeRoo 1 National Human Genome Research Institute, National Institutes of Health , Bethesda, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Malak Abbas 2 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gabriel Goodney 3 National Heart Lung and Blood Institute, National Institutes of Health , Bethesda, MD, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Merry L. Lindsey 4 Department of Biomedical Sciences, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ashton F. Oliver 5 Department of Integrative Genomics and Epidemiology, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Han Le 5 Department of Integrative Genomics and Epidemiology, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Antwi-Boasiako Oteng 5 Department of Integrative Genomics and Epidemiology, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amadou Gaye 5 Department of Integrative Genomics and Epidemiology, Meharry Medical College , Nashville, TN, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amadou Gaye For correspondence: amadou.gaye{at}mmc.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background Hypertension is a leading contributor to cardiovascular disease in reproductive-age women, with African American women experiencing disproportionate risk and severity. Oral contraceptive (OC) use has been implicated in elevating blood pressure, but the underlying molecular mechanisms remain unclear. Understanding the proteomic alterations associated with OC use may provide novel insights into the biological pathways contributing to OC-related hypertensive risk. Objective To identify proteomic signatures associated with oral contraceptive use among premenopausal African American women with stage 2 hypertension and elucidate pathways that may contribute to blood pressure regulation in this population. Study Design We evaluated 2,941 serum proteins measured on the Olink platform to assess associations with oral contraception use in 51 pre-menopausal women with untreated stage 2 hypertension (13 OC users and 38 non-users). A generalized linear model was fitted for each protein, adjusting for age, body mass index, alcohol consumption, level of education, and physical activity. Pathway enrichment analysis was carried out to identify common pathways among the proteins associated with oral contraceptive use. Gene Ontology enrichment analysis was conducted to gain insight into the functional characteristics of the proteins and the underlying biology. Results We identified 44 proteins significantly associated with OC use (FDR ≤ 0.05), including known and novel candidates. Among these, 13 proteins were elevated in OC users, including SERPINA6, SERPINA7, and TNFSF13, markers implicated in hormone transport, RAAS activation, and chronic inflammation. Proteins such as PLAU and PLAUR were enriched in sympathetic nervous system signaling and coagulation pathways, while SCG3 suggested neuroendocrine involvement. Enrichment analysis revealed overrepresentation of pathways related to complement and coagulation cascades, cholesterol metabolism, and the renin–angiotensin–aldosterone system.. Conclusions OC use among premenopausal African American women, with stage 2 untreated hypertension, is associated with alterations in circulating proteins involved in neurohormonal signaling, immune modulation, lipid regulation, and vascular remodeling. The findings provide novel insights into the proteomic landscape associated with oral contraceptive in these women. INTRODUCTION Hypertension remains a major public health burden and a key contributor to cardiovascular disease (CVD) mortality globally 1 . Sex modulates both CVD risk and individual response to treatment, and understanding sex-specific differences in the pathophysiology of CVD is a necessary step for personalized medicine. 2 In a nationally representative 2011-2016 survey conducted in non-pregnant women of reproductive age, 9.3% had hypertension; among those, 16.9% were unaware that they had hypertension and over 40% had uncontrolled hypertension. 3 Cumulative exposure to elevated blood pressure (BP) over time is associated with increased risk of CVD outcomes and mortality, above and beyond current BP status. This suggests the importance of a life course approach to hypertension, with accumulated years of elevated BP in early to mid-adulthood having longer term implications for CVD risk. 4 There are significant racial disparities for African Americans compared to Whites in prevalence, severity, and control of hypertension. African American women have a higher age-adjusted prevalence of hypertension (56.7%) than non-Hispanic White women (36.7%) 5 and tend to develop hypertension at earlier stages of life and endure more severe manifestations of the condition. 6 Among younger, premenopausal women with hypertension, African Americans were over two times more likely to have uncontrolled BP than whites. These disparities persist despite similar or even greater health awareness and healthcare engagement, underscoring the need to better understand context-specific risk factors in this population 5 . One such context is the use of OCs, a common hormonal method of birth control used by millions of women worldwide. Although generally safe, OC use has been associated with modest increases in BP and elevated CVD risk, particularly in women with pre-existing hypertension or additional risk factors such as smoking, obesity, or family history of CVD 6 - 8 . Notably, women with stage 2 hypertension are often excluded from prospective trials of hormonal contraception, resulting in major evidence gaps regarding the biological effects of OCs in this vulnerable group 9 . Previous work has suggested that OC-associated hypertension may involve activation of the renin– angiotensin–aldosterone system (RAAS), increased sympathetic nervous system activity, and altered regulation of antidiuretic hormone (ADH) 9 - 11 . Despite these known physiological effects, few studies have systematically profiled the circulating proteome to identify molecular changes linked to OC use in hypertensive women. Among healthy, reproductive-aged women with a low absolute risk for CVD, the small increase in BP due to OC use is unlikely to be clinically relevant. However, for women with multiple risk factors (e.g., age > 35, high blood pressure), OC-use may increase CVD risk to an unacceptable level. In this study, we sought to identify proteomic alterations associated with OC use in premenopausal African American women with stage 2 hypertension, a group at elevated risk for hypertensive complications yet underrepresented in biomedical research. Our aim is to (1) characterize the proteomic signature of OC use in this population and (2) explore the biological relevance of identified proteins in the context of known hypertension mechanisms. MATERIAL AND METHODS Study population We used data from the GENomics, Environmental FactORs, and the Social DEterminants of Cardiovascular Disease in African Americans STudy (GENE-FORECAST), a study of 669 self-identified, U.S.-born African American men and women aged 21 to 65 years, recruited from the metropolitan Washington D.C. area during 2014-2022. The study protocol was approved by the Institutional Review Board of the U.S. National Institutes of Health (NIH), and written informed consent was provided by all participants. Data collection took place during a visit to the NIH Clinical Research Center. Pregnancy was an exclusion criterion for the study, and women were asked about menopausal status before undergoing a mandatory pregnancy test. Participants provided blood samples, underwent a physical exam and filled out questionnaires on medical history and health behaviors. BP measurements were taken in triplicate and averaged. OC use was obtained in a questionnaire aimed at collecting a comprehensive list of current medications. GENE-FORECAST utilized a multi-omics systems biology approach, facilitating comprehensive, multi-dimensional characterization of health and disease among African Americans. Here, we isolated the differences in protein profiles for OC use among African American women with uncontrolled hypertension. Our inclusion criteria were women of pre-menopausal status with stage 2 hypertension (defined as systolic BP ≥ 140 mm HG or diastolic BP ≥ 90 mm HG based on the average of clinic measurements) who were not taking anti-hypertensive medication. Proteomic data The ethylenediamine tetraacetic acid (EDTA) plasma samples from the GENE-FORECAST were sent to the Olink Proteomics Analysis Service in Boston, USA. All proteomic analyses were conducted collectively as one single batch. The Explore 3072 assay, utilizing eight Explore 384 Olink panels (Cardiometabolic, Cardiometabolic II, Inflammation, Inflammation II, Neurology, Neurology II, Oncology, Oncology II), were run to assess relative expression of a total of 2,947 unique proteins. Proximity Extension Assay echnology was conducted according to the Olink AB manufacturer procedures by the certified laboratory. Briefly, the technique relies on the use of antibodies labelled with unique DNA oligonucleotides that bind to their target protein present in the sample. The DNA oligonucleotides, when in proximity on a target protein, undergo hybridization and act as a template for DNA polymerase-dependent extension, forming a unique double-stranded DNA barcode proportionate to the initial protein concentration. Quantification of resulting DNA amplicons is accomplished through high-throughput DNA sequencing, generating a digital signal reflective of the number of DNA hybridization events corresponding to the protein concentration in the original sample. The measurement of protein levels is based on Normalized Protein eXpression (NPX) values, serving as a relative protein quantification unit. This quantification is normalized to account for systematic noise arising from sample processing and technical variation, leveraging internal controls and sample controls. NPX units are on a log2 scale, where a one NPX unit increase indicates a two-fold rise in the concentration of amplicons representing the target protein compared to the internal control. A total of 2,941 of the 2,947 proteins (99.8%) passed quality controls and were included in the analysis. Statistical analysis Separate generalized linear models were fit for each of the 2,941 proteins, with protein level set as the outcome variable and main exposure of interest set as oral contraceptive use (no=0; yes=1), adjusting for age, body mass index (BMI), physical activity level, and education level. The p-values of the associations were adjusted for false discovery rate (FDR) using the Benjamini & Hochberg (BH) approach, with an FDR adjusted p-value ≤ 0.05 considered statistically significant. Pathway enrichment analysis was undertaken to identify molecular pathways enriched in the set of proteins found to be associated with OC use. This analysis was carried out with the R algorithm pathfinR , 10 a tool that defines subnetworks in a cluster of genes and identifies pathways over-represented in those subnetworks. Pathways with a BH adjusted enrichment p-value <0.05 were considered significantly overrepresented. Gene Ontology (GO) enrichment analyses were subsequently conducted with the set of proteins associated with OC use to provide a systematic framework for annotating and deciphering biological functions, processes, and cellular components linked with the proteins and to gain insight into their roles within cellular and organismal contexts. GO analysis were conducted using the R libraries ‘enrichGO’ which runs a hypergeometric test to determine whether the set of proteins associated with OCs is enriched for specific GO terms compared to what would be expected by chance. GO terms with a BH adjusted enrichment p-value <0.05 were considered significantly overrepresented. RESULTS Our study sample included 51 pre-menopausal women with stage 2 hypertension; 13 OC users and 38 non-users as after excluding 638 for reasons outlined in Figure 1 . The 51 samples are decribed in Table 1 . As expected, the two groups of OC users and non-users were similar in mean age (35 and 38 years, respectively) and mean BP (181 and 180 mmHg Systolic BP, respectively, and 72 mmHg Diastolic BP). Nearly all subjects were non-smokers. OC users were less likely to have a healthy BMI and more likely to drink alcohol, be a college graduate, and engage in moderate or greater than moderate physical activity than non-users. View this table: View inline View popup Download powerpoint Table 1: Baseline characteristics of the 51 women included in the analysis. Download figure Open in new tab Figure 1: Flowchart illustrating sample selection: of 669 pre-menopausal women screened, 618 were excluded based on criteria detailed in the diagram, resulting in a final analytic sample of 51 women Download figure Open in new tab Figure 2: Proteins linked to hormonal contraceptive use, with beta coefficients and corresponding 95% confidence intervals and adjusted p-values. The genes are listed from negative to positive beta coefficient. A total of 2,941 (99.8%) out of 2,947 proteins passed quality control. All the measurements were conducted in a single, centralized batch ensuring minimal batch effects and high internal consistency. The technology employed by Olink offers high specificity and sensitivity by relying on dual antibody recognition and DNA barcoding, reducing cross-reactivity and improving signal fidelity. Built-in internal and external controls enabled stringent normalization and correction for technical variation. Proteins Associated with OC Use We identified 44 proteins significantly associated with oral contraceptive use ( Figure 1 and Supplemental Table T1). There were 31 proteins exhibiting lower levels among OC users and 13 proteins with higher levels. We identified 44 proteins significantly associated with OC use; the results are summarized graphically in Figure 1 an detailed in Supplemental Table T1. Of these 44 proteins, 31 were found at lower levels and 13 at higher levels among OC users. Notably, several proteins previously implicated in blood pressure regulation and hormonal response pathways were differentially expressed. Elevated levels of SERPINA6 and SERPINA7, key hormone-binding proteins, point toward activation of the renin–angiotensin–aldosterone system (RAAS). Similarly, higher expression of TNFSF13, involved in inflammatory signaling, and SCG3, a neuroendocrine granin protein, supports a role for inflammation and sympathetic nervous system activation in OC-associated hypertension. We also observed increases in PLAU and PLAUR, which contribute to both coagulation and neurovascular signaling. Conversely, proteins such as ANGPTL3 and NOTCH3, involved in lipid metabolism and vascular remodeling respectively, were downregulated in OC users, suggesting additional pathways of vascular and metabolic dysregulation. Novel findings include differential expression of SMAD5, potentially linking OC use to TGF-β/BMP-mediated vascular remodeling. Pathway Enrichment Analysis Pathway enrichment analysis of the 44 differentially expressed proteins revealed multiple significantly enriched biological pathways (FDR ≤ 0.05), outlined in Figure 3 . The most significant pathway was complement and coagulation cascades (adjusted p = 2×10 −5 ), supported by the involvement of PLAU, PLAUR, and PLG, which are key regulators of fibrinolysis and vascular homeostasis. Additional enriched pathways included prostate cancer, proteoglycans in cancer, and cholesterol metabolism, involving proteins like ANGPTL3 and LPA. Immune and metabolic signaling pathways were also highlighted, including TGF-beta signaling (SMAD5), Notch signaling (NOTCH3), and PPAR signaling. Download figure Open in new tab Figure 3: Pathways enriched in the set 44 proteins associated with hormonal contraceptive use, ranked by decreasing p-value of enrichment. Several pathways related to inflammation and cellular communication such as Th1 and Th2 cell differentiation, ECM–receptor interaction, gap junctions, and cell adhesion molecules, were also enriched. Notably, a number of cancer-associated pathways (thyroid cancer, melanoma, non-small cell lung cancer) reached significance, potentially reflecting shared molecular mediators of cell proliferation and vascular remodeling. The list of significantly enriched pathways, including the adjusted p-values and the proteins associated with oral contraceptive use that contribute to each pathway, is provided in Supplemental Table T1. Gene Ontology Enrichment Analysis Gene Ontology enrichment analysis revealed 25 GO terms significantly enriched biological processes and molecular functions ( Figure 4 ) involving 39 of the 44 proteins associated with OC use. Notably, enriched categories include peptidase regulator activity, endopeptidase inhibitor activity, and serine-type endopeptidase inhibitor activity, which prominently feature proteins like SERPINA6, SERPINA7, FETUB, SPINK1, and PZP, all of which were elevated in OC users. These categories highlight functional roles related to hormone transport, protease regulation, and extracellular matrix remodeling. Download figure Open in new tab Figure 4: Gene Ontology terms enriched in the set 44 proteins associated with hormonal contraceptive use, ranked by decreasing p-value of enrichment. Other enriched GO terms include complement activation and humoral immune response, with involvement of proteins such as PLG, ITIH3, and SERPINA6, pointing to immune-modulatory processes that may intersect with vascular and coagulation pathways. Additionally, categories related to lipoprotein particle remodeling and cholesterol efflux were also enriched, involving PLTP, ANGPTL3, and LPA, decreased in OC users. The full list of enriched GO terms, adjusted p-values, and contributing proteins is provided in Supplemental Table T2, which includes all significantly enriched pathways and the subset of OC-associated proteins linked to each. DISCUSSIONS The aim of this study was to identify proteomic alterations associated with oral contraceptive use in premenopausal African American women with stage 2 hypertension. Three key findings emerged. First, we identified 44 proteins significantly associated with OC use, encompassing both previously reported markers and novel candidates. Second, our results provide mechanistic support for established pathways of OC-induced hypertension, including activation of RAAS, heightened sympathetic nervous system activity, and altered regulation of antidiuretic hormone. Third, we highlight specific proteins such as SERPINA6, SERPINA7, and TNFSF13, that offer new insights into how OCs may influence cardiovascular risk in hypertensive women. Collectively, these findings suggest that OC use contributes to hypertension through multiple, interconnected molecular pathways. One of the most important findings was the identification of proteins involved in the RAAS pathway, such as SERPINA6 (corticosteroid-binding globulin) and SERPINA7 (thyroxine-binding globulin), which were elevated in OC users. These proteins play important roles in hormone transport and may influence RAAS components, contributing to increased sodium and water retention and elevated BP 11 , 12 . Gene Ontology analysis further supports their involvement, highlighting enrichment in peptidase inhibitor activity, particularly among serpin family proteins which regulate protease activity essential for vascular stability and inflammatory control 13 , 14 . Additionally, TNFSF13 and TNFSF10, both associated with chronic inflammation, are consistent with the hypothesis that impaired RAAS feedback inhibition exacerbates hypertension in OC users 15 , 16 . These findings provide new insights into the inflammatory mechanisms linking OC use to BP regulation. We also observed increased levels of SCG3 (secretogranin III), PLAU (plasminogen activator urokinase), and PLAUR (plasminogen activator urokinase receptor), supporting the hypothesis that OCs enhance sympathetic nervous system activity, which contributes to elevated BP. Pathway enrichment identified PLAU and PLAUR as part of the complement and coagulation cascades, suggesting that these proteins may play dual roles in sympathetic signaling and promoting a prothrombotic state in OC users 17 , 18 . PLAU and PLAUR are expressed in sympathetic neurons and amplify neurogenic vascular responses and neurotransmitter release 19 - 21 , thereby influencing vascular tone and BP regulation independently of plasmin generation. Although SCG3 is less directly studied in this context, related granin proteins such as chromogranin A are established modulators of catecholamine secretion and sympathetic outflow, suggesting a plausible role for SCG3 in OC-associated neuroendocrine activation 22 , 23 . Collectively, these proteins are involved in neuroendocrine secretion, tissue remodeling, and inflammation, processes central to sympathetic tone and vascular function. Furthermore, the changes in LPA and COL6A3 levels suggest that OCs may alter ADH regulation, thereby influencing fluid balance and extracellular matrix integrity, both important components of BP homeostasis. In parallel, LPA, along with PLTP and ANGPTL3, showed patterns consistent with OC-induced dyslipidemia, which may further impact vascular tone and inflammatory status 24 . LPA induces vasoconstriction and stimulates intracellular calcium signaling in vascular smooth muscle cells, mechanisms known to trigger ADH release under osmotic and volume stress conditions 25 . COL6A3, a structural ECM protein expressed in the kidney interstitium, is influenced by hormonal status and may alter renal responsiveness to ADH by modulating water reabsorption capacity and tubular architecture 26 - 28 . Notably, ANGPTL3, a regulator of lipid metabolism, was decreased in OC users, suggesting complex interactions between OCs, lipid regulation, and hypertension 29 , 30 . In addition, NOTCH3, a receptor involved in vascular smooth muscle cell fate and remodeling, was differentially expressed and may act downstream of inflammatory and lipid-related signaling to influence arterial pressure regulation and vascular adaptation to hormonal exposure 31 . Several of the proteins identified, including PZP, SERPINA6, FETUB, PLG, PGLYRP2, and ITIH3, have been reported previously in large-scale population-based proteomic studies, such as the CHRIS and NESDA cohorts 32 , 33 . The consistency in direction and magnitude of associations for proteins such as PZP, SERPINA6 (linked to RAAS activation), FETUB, PLG (coagulation), PGLYRP2, and ITIH3 adds confidence to our findings. At the same time, we uncovered novel protein associations not previously linked to OC use. Notably, SMAD5, a receptor-regulated SMAD in the TGF-β/BMP signaling axis, emerged as a new candidate 34 . SMAD5 mediates endothelial–mesenchymal transition and promotes extracellular matrix deposition and vascular smooth muscle proliferation, processes that are key to vascular remodeling and fibrosis in hypertension models 35 , 36 . These results suggest that hormonal modulation of the TGF-β/BMP–SMAD5 signaling axis may represent one mechanism through which OCs contribute to vascular remodeling and elevated blood pressure 8 . Taken together, these findings may help inform future efforts in CVD-related biomarker discovery and personalized contraceptive counseling for women with hypertension 37 . A precision medicine approach, informed by proteomic profiling, could enable safer contraceptive choices that minimize cardiovascular risk in high-risk populations 37 . Our study has limitations. We did not collect data on OC formulation, dose, or duration, which limits assessment of specific OC types. The cross-sectional design precludes causal inference. Although we adjusted for important covariates, residual confounding cannot be ruled out. Finally, while most participants used pill-based OCs, potential misclassification of other hormonal contraceptive use may have diluted our estimates. In conclusion, OC use in African American women with stage 2 hypertension is associated with proteomic changes across multiple pathways related to blood pressure regulation. Our findings confirm previously reported protein alterations while also identifying new candidate biomarkers and mechanisms that merit further investigation. This work underscores the importance of considering molecular heterogeneity in contraceptive risk and supports more individualized approaches to reproductive and cardiovascular care. ACKNOWLEDGEMENTS This work was supported by the Chan Zuckerberg Initiative’s Foundation for Accelerate Precision Health Program to Advance Genomics Research at Meharry Medical College (CZIF2022-007043); by the National Institute of Health under award numbers EY033264, GM151274, UC2MD019626; and by the Veterans Affairs Office of Research and Development under award number CX002780. Dr Ayo Priscille Doumatey, National Institutes of Health – National Human Genome Research Institutes. Funder Information Declared Chan Zuckerberg Initiative (United States), https://ror.org/02qenvm24 , CZIF2022-007043 National Institutes of Health Clinical Center , EY033264 , GM151274 , UC2MD019626 United States Department of Veterans Affairs, https://ror.org/05rsv9s98 , CX002780 Footnotes Conflict of Interest The authors report no conflict of interest. A complete reinterpretation of the results, a revised framing of the hypothesis, and a significant refocusing of the introduction on hypertension. The following improvements have been made: - Rewritten the introduction to remain tightly focused on hypertension, removing tangential elements and better grounding the study in the context of hypertensive disorders in reproductive-age women. - Revised the hypothesis statement for clarity and fully restructured the results and discussion sections to reflect the new interpretation of findings. - Clarified the derivation of the analytic sample, providing a transparent account of how we arrived at 51 participants from an initial cohort of 669. This is now clearly outlined in the text and visually represented in a new figure. REFERENCES 1. ↵ Mills KT , Stefanescu A , He J. The global epidemiology of hypertension . Nat Rev Nephrol 2020 ; 16 ( 4 ): 223 – 237 . DOI: 10.1038/s41581-019-0244-2 . OpenUrl CrossRef PubMed 2. ↵ Baetta R , Pontremoli M , Martinez Fernandez A , Spickett CM , Banfi C. Proteomics in cardiovascular diseases: Unveiling sex and gender differences in the era of precision medicine . J Proteomics 2018 ; 173 : 62 – 76 . DOI: 10.1016/j.jprot.2017.11.012 . OpenUrl CrossRef PubMed 3. ↵ Azeez O , Kulkarni A , Kuklina EV , Kim SY , Cox S. Hypertension and Diabetes in Non-Pregnant Women of Reproductive Age in the United States . Prev Chronic Dis 2019 ; 16 : E146 . DOI: 10.5888/pcd16.190105 . OpenUrl CrossRef PubMed 4. ↵ Reges O , Ning H , Wilkins JT , et al. Association of Cumulative Systolic Blood Pressure With Long-Term Risk of Cardiovascular Disease and Healthy Longevity: Findings From the Lifetime Risk Pooling Project Cohorts . Hypertension 2021 ; 77 ( 2 ): 347 – 356 . DOI: 10.1161/HYPERTENSIONAHA.120.15650 . OpenUrl CrossRef 5. ↵ Ostchega Y , Fryar CD , Nwankwo T , Nguyen DT . Hypertension Prevalence Among Adults Aged 18 and Over: United States, 2017-2018 . NCHS Data Brief 2020 ( 364 ): 1 – 8 . ( https://www.ncbi.nlm.nih.gov/pubmed/32487290 ). 6. ↵ Ferdinand KC , Armani AM . The management of hypertension in African Americans . Crit Pathw Cardiol 2007 ; 6 ( 2 ): 67 – 71 . DOI: 10.1097/HPC.0b013e318053da59 . OpenUrl CrossRef PubMed 7. Meyerovitz CV , Juraschek SP , Ayturk D , et al. Social Determinants, Blood Pressure Control, and Racial Inequities in Childbearing Age Women With Hypertension, 2001 to 2018 . J Am Heart Assoc 2023 ; 12 ( 5 ): e027169 . DOI: 10.1161/JAHA.122.027169 . OpenUrl CrossRef PubMed 8. ↵ Cameron NA , Blyler CA , Bello NA . Oral Contraceptive Pills and Hypertension: A Review of Current Evidence and Recommendations . Hypertension 2023 ; 80 ( 5 ): 924 – 935 . DOI: 10.1161/HYPERTENSIONAHA.122.20018 . OpenUrl CrossRef 9. ↵ Farley TM , Collins J , Schlesselman JJ . Hormonal contraception and risk of cardiovascular disease . An international perspective. Contraception 1998 ; 57 ( 3 ): 211 – 30 . DOI: 10.1016/s0010-7824(98)00019-5 . OpenUrl CrossRef PubMed 10. ↵ Ulgen E , Ozisik O , Sezerman OU . pathfindR: An R Package for Comprehensive Identification of Enriched Pathways in Omics Data Through Active Subnetworks . Front Genet 2019 ; 10 : 858 . DOI: 10.3389/fgene.2019.00858 . OpenUrl CrossRef PubMed 11. ↵ Crislip GR , Johnston JG , Douma LG , et al. Circadian Rhythm Effects on the Molecular Regulation of Physiological Systems . Compr Physiol 2021 ; 12 ( 1 ): 2769 – 2798 . DOI: 10.1002/cphy.c210011 . OpenUrl CrossRef PubMed 12. ↵ Crawford AA , Bankier S , Altmaier E , et al. Variation in the SERPINA6/SERPINA1 locus alters morning plasma cortisol, hepatic corticosteroid binding globulin expression, gene expression in peripheral tissues, and risk of cardiovascular disease . J Hum Genet 2021 ; 66 ( 6 ): 625 – 636 . DOI: 10.1038/s10038-020-00895-6 . OpenUrl CrossRef 13. ↵ Kelly-Robinson GA , Reihill JA , Lundy FT , et al. The Serpin Superfamily and Their Role in the Regulation and Dysfunction of Serine Protease Activity in COPD and Other Chronic Lung Diseases . Int J Mol Sci 2021 ; 22 ( 12 ). DOI: 10.3390/ijms22126351 . OpenUrl CrossRef 14. ↵ Kryvalap Y , Czyzyk J. The Role of Proteases and Serpin Protease Inhibitors in beta-Cell Biology and Diabetes . Biomolecules 2022 ; 12 ( 1 ). DOI: 10.3390/biom12010067 . OpenUrl CrossRef 15. ↵ Krishnan A , Katsumi T , Guicciardi ME , et al. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Deficiency Promotes the Ductular Reaction, Macrophage Accumulation, and Hepatic Fibrosis in the Abcb4(-/-) Mouse . Am J Pathol 2020 ; 190 ( 6 ): 1284 – 1297 . DOI: 10.1016/j.ajpath.2020.02.013 . OpenUrl CrossRef PubMed 16. ↵ Fountain JH , Kaur J , Lappin SL . Physiology, Renin Angiotensin System . StatPearls . Treasure Island (FL ) 2025 . 17. ↵ Travers RJ , Smith SA , Morrissey JH . Polyphosphate, platelets, and coagulation . Int J Lab Hematol 2015 ; 37 Suppl 1 (0 1): 31 – 5 . DOI: 10.1111/ijlh.12349 . OpenUrl CrossRef PubMed 18. ↵ Lopez-Pedrera C , Jardi M , del Mar Malagon M , et al. Tissue factor (TF) and urokinase plasminogen activator receptor (uPAR) and bleeding complications in leukemic patients . Thromb Haemost 1997 ; 77 ( 1 ): 62 – 70 . ( https://www.ncbi.nlm.nih.gov/pubmed/9031451 ). OpenUrl PubMed 19. ↵ Schaefer U , Machida T , Vorlova S , Strickland S , Levi R. The plasminogen activator system modulates sympathetic nerve function . J Exp Med 2006 ; 203 ( 9 ): 2191 – 200 . DOI: 10.1084/jem.20060077 . OpenUrl Abstract / FREE Full Text 20. Primak A , Bozov K , Rubina K , et al. Morphogenetic theory of mental and cognitive disorders: the role of neurotrophic and guidance molecules . Front Mol Neurosci 2024 ; 17 : 1361764 . DOI: 10.3389/fnmol.2024.1361764 . OpenUrl CrossRef PubMed 21. ↵ Meng J , Chen W , Wang J. Interventions in the B-type natriuretic peptide signalling pathway as a means of controlling chronic itch . Br J Pharmacol 2020 ; 177 ( 5 ): 1025 – 1040 . DOI: 10.1111/bph.14952 . OpenUrl CrossRef PubMed 22. ↵ Bourebaba Y , Mularczyk M , Marycz K , Bourebaba L. Catestatin peptide of chromogranin A as a potential new target for several risk factors management in the course of metabolic syndrome . Biomed Pharmacother 2021 ; 134 : 111113 . DOI: 10.1016/j.biopha.2020.111113 . OpenUrl CrossRef PubMed 23. ↵ Yuan CY , Zuo L , Dong YC , Liu BX , Qi H. Secretogranin III: a promising therapeutic target for intraocular neovascular lesions . Int Ophthalmol 2025 ; 45 ( 1 ): 26 . DOI: 10.1007/s10792-024-03393-2 . OpenUrl CrossRef PubMed 24. ↵ Turki A , Ayalew A , Mossie A , Mitiku S. Effects of hormonal contraceptives on lipid profile among women attending family planning unit in Goba Town Public Health Facilities, Bale, Southeast Ethiopia: a comparative cross-sectional study . Reprod Health 2023 ; 20 ( 1 ): 185 . DOI: 10.1186/s12978-023-01727-4 . OpenUrl CrossRef PubMed 25. ↵ Ven K , Besztercei B , Janovicz A , et al. LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas . Int J Mol Sci 2024 ; 25 ( 13 ). DOI: 10.3390/ijms25136872 . OpenUrl CrossRef 26. ↵ Cheng J , Li G , Wang W , Stovall DB , Sui G , Li D. Circular RNAs with protein-coding ability in oncogenesis . Biochim Biophys Acta Rev Cancer 2023 ; 1878 ( 4 ): 188909 . DOI: 10.1016/j.bbcan.2023.188909 . OpenUrl CrossRef 27. Su L , Sun Q , Li Y , et al. Collagen V regulates renal function after kidney injury and can be pharmacologically targeted to enhance kidney repair in mice . Sci Transl Med 2025 ; 17 ( 793 ): eads7714 . DOI: 10.1126/scitranslmed.ads7714 . OpenUrl CrossRef PubMed 28. ↵ Karamanos NK , Theocharis AD , Piperigkou Z , et al. A guide to the composition and functions of the extracellular matrix . FEBS J 2021 ; 288 ( 24 ): 6850 – 6912 . DOI: 10.1111/febs.15776 . OpenUrl CrossRef PubMed 29. ↵ Sylvers-Davie KL , Davies BSJ . Regulation of lipoprotein metabolism by ANGPTL3, ANGPTL4, and ANGPTL8 . Am J Physiol Endocrinol Metab 2021 ; 321 ( 4 ): E493 – E508 . DOI: 10.1152/ajpendo.00195.2021 . OpenUrl CrossRef PubMed 30. ↵ Stitziel NO , Khera AV , Wang X , et al. ANGPTL3 Deficiency and Protection Against Coronary Artery Disease . J Am Coll Cardiol 2017 ; 69 ( 16 ): 2054 – 2063 . DOI: 10.1016/j.jacc.2017.02.030 . OpenUrl FREE Full Text 31. ↵ Morris HE , Neves KB , Montezano AC , MacLean MR , Touyz RM . Notch3 signalling and vascular remodelling in pulmonary arterial hypertension . Clin Sci (Lond) 2019 ; 133 ( 24 ): 2481 – 2498 . DOI: 10.1042/CS20190835 . OpenUrl CrossRef PubMed 32. ↵ Ramsey JM , Cooper JD , Penninx BW , Bahn S. Variation in serum biomarkers with sex and female hormonal status: implications for clinical tests . Sci Rep 2016 ; 6 : 26947 . DOI: 10.1038/srep26947 . OpenUrl CrossRef PubMed 33. ↵ Dordevic N , Dierks C , Hantikainen E , et al. Pervasive Influence of Hormonal Contraceptives on the Human Plasma Proteome in a Broad Population Study . medRxiv 2023 :2023.10.11.23296871. DOI: 10.1101/2023.10.11.23296871 . OpenUrl Abstract / FREE Full Text 34. ↵ Lin Z , Zhuang J , He L , et al. Exploring Smad5: a review to pave the way for a deeper understanding of the pathobiology of common respiratory diseases . Mol Med 2024 ; 30 ( 1 ): 225 . DOI: 10.1186/s10020-024-00961-1 . OpenUrl CrossRef PubMed 35. ↵ Qian C , Dong G , Yang C , et al. Broadening horizons: molecular mechanisms and disease implications of endothelial-to-mesenchymal transition . Cell Commun Signal 2025 ; 23 ( 1 ): 16 . DOI: 10.1186/s12964-025-02028-y . OpenUrl CrossRef 36. ↵ Ma J , Sanchez-Duffhues G , Goumans MJ , Ten Dijke P. TGF-beta-Induced Endothelial to Mesenchymal Transition in Disease and Tissue Engineering . Front Cell Dev Biol 2020 ; 8 : 260 . DOI: 10.3389/fcell.2020.00260 . OpenUrl CrossRef PubMed 37. ↵ De Leo V , Musacchio MC , Cappelli V , Piomboni P , Morgante G. Hormonal contraceptives: pharmacology tailored to women’s health . Hum Reprod Update 2016 ; 22 ( 5 ): 634 – 46 . DOI: 10.1093/humupd/dmw016 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted January 28, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Proteomic Alterations Associated with Oral Contraceptive Use in Hypertensive Premenopausal African American Women Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Proteomic Alterations Associated with Oral Contraceptive Use in Hypertensive Premenopausal African American Women Lisa DeRoo , Malak Abbas , Gabriel Goodney , Merry L. Lindsey , Ashton F. Oliver , Han Le , Antwi-Boasiako Oteng , Amadou Gaye bioRxiv 2024.08.12.607634; doi: https://doi.org/10.1101/2024.08.12.607634 Share This Article: Copy Citation Tools Proteomic Alterations Associated with Oral Contraceptive Use in Hypertensive Premenopausal African American Women Lisa DeRoo , Malak Abbas , Gabriel Goodney , Merry L. Lindsey , Ashton F. Oliver , Han Le , Antwi-Boasiako Oteng , Amadou Gaye bioRxiv 2024.08.12.607634; doi: https://doi.org/10.1101/2024.08.12.607634 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genomics Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17728) Bioengineering (13916) Bioinformatics (42037) Biophysics (21489) Cancer Biology (18637) Cell Biology (25553) Clinical Trials (138) Developmental Biology (13401) Ecology (19941) Epidemiology (2067) Evolutionary Biology (24367) Genetics (15622) Genomics (22547) Immunology (17764) Microbiology (40475) Molecular Biology (17208) Neuroscience (88747) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9835) Zoology (2272)