Genistein for the Reduction of Adverse Cardiovascular Events (GRACE)
Genistein for the Reduction of Adverse Cardiovascular Events (GRACE)
People living with HIV (PLHIV) live longer because of modern HIV medicines, but even with these treatments, their bodies are more inflamed. Long term inflammation increases the risk of heart disease, chronic pain, lung disease, dementia, arthritis, and other infections. Because some anti-inflammatory medicines can cause serious side effects, interact with HIV drugs, or be costly, safer and more affordable treatment options are needed. Genistein is a natural compound found in soy foods. It is considered safe and has been shown to reduce inflammation and improve blood vessel health. This study will test whether genistein can lower inflammation in the blood, improve blood vessel health, and reduce pain in PLHIV.
A. Rationale Despite the gains in life expectancy afforded by effective antiretroviral therapy (ART), people living with HIV (PLHIV) continue to exhibit persistent immune activation, immune dysfunction and chronic inflammation, all of which contribute to an elevated risk of cardiovascular disease (CVD) [1-5]. To address this excess risk, there is an ongoing need for safe adjunctive therapies capable of reducing inflammation, particularly because conventional anti-inflammatory agents can cause adverse effects or interact with ART [6-9].
. B. Background CVD in PLHIV on ART The World Health Organization estimates that there are 41 million PLHIV with 32 million (~77%) receiving ART and an additional 1.5 million more cases predicted to arise each year (https://www.who.int/teams/global-hiv-hepatitis-and-stis-programmes/hiv/strategic-information/hiv-data-and-statistics). PLHIV on ART represent a state of virologic control without full immunologic or inflammatory normalization, characterized by persistent immune activation, chronic inflammation, and multisystem dysregulation all contributing to increased risk of CVD [1-5, 10-14]. This excess risk is not fully explained by traditional CVD risk factors, such as hypertension, dyslipidemia, diabetes, smoking, obesity, sedentary lifestyle, unhealthy diet and psychosocial stress. Additionally, ARTs themselves are attributed to increased risk of hypertension, dyslipidemia and diabetes [2]. Adding to this complexity, the treatment of dyslipidemia with statins--that have the added benefits of pleiotropic anti-inflammatory off-targets--does not reduce the CVD risk to levels to those of an unaffected population [2] and may increase the incidence of diabetes [7, 15], myalgia, muscle weakness or myopathy [7].
In PLHIV, paramount among non-traditional CVD risk factors is chronic low grade systemic inflammation, characterized by persistently elevated inflammatory and immune activation markers, notably IL 6, TNF α, IL 1β, C reactive protein, D dimer, sCD14, and sCD163 [3, 16, 17]. These abnormalities are driven by multiple overlapping mechanisms, including residual HIV transcription, gut associated tissue damage with microbial translocation, chronic co infections, and incomplete immune reconstitution [18]. Gut barrier dysfunction triggered by early HIV is hypothesized to be the potential underlying cause for ongoing immune dysfunction despite effective ARTs (Fig 1). Growing evidence that gut microbiome associated metabolites regulate gene expression profiles of immune cells linked to inflammation [19]. Sustained immune activation of T cells (e.g., CD38⁺HLA DR⁺), monocytes, and innate immune pathways persists despite undetectable viral load [20]. Immune dysregulation remains, accompanied by reduced naïve T cell pools, immune senescence, and immune exhaustion. Persistent monocyte and macrophage activation has been directly linked to coronary plaque burden and vascular inflammation, reinforcing the connection between immune activation and CVD [1, 2].
These inflammatory and immune abnormalities converge on endothelial dysfunction, a central and early event in CVD pathogenesis. In PLHIV, chronic inflammation and ART exposure impair nitric oxide signaling, increase oxidative stress, and promote arterial stiffness [13]. Endothelial dysfunction is further exacerbated by comorbid exposures and conditions common in this population. Cannabis use, for example, is four times more prevalent among PLHIV [21] and has been associated with vascular inflammation, oxidative stress, and impaired endothelial dependent vasodilation [22-24]. Chronic pain states, in an estimated 25% to 80% of PLHIV on ART, similarly contribute through sustained sympathetic activation and neuroimmune signaling, which impair vascular repair mechanisms [25]. Collectively, these interrelated mechanisms drive the increased burden of CVD and other non AIDS comorbidities in PLHIV receiving ART.
Genistein as a therapeutic for PLHIV at risk of CVD Non immunosuppressive adjunctive therapeutic strategies that safely target upstream inflammation and endothelial dysfunction represent a critical unmet need to reduce CVD in PLHIV. Genistein, a naturally occurring soy derived isoflavone consumed in human diets for millennia, exhibits multi modal anti inflammatory, antioxidant, and vasoprotective properties (Fig 2) [26]. Mechanistically, genistein inhibits key pathways activated in treated HIV: NF κB-dependent transcription, pro inflammatory cytokine signaling (Fig 3), inducible nitric oxide synthase activity, and reactive oxygen species generation. Importantly, genistein does not induce global immune suppression and has demonstrated a favorable safety profile.
Anti-Inflammatory properties of genistein Inflammation, while normally a protective biological response triggered by harmful stimuli, can become harmful when persistent and/or systemic.
Inhibition of NF κB signaling. At the molecular level, genistein inhibits NF κB primarily by suppressing IκB kinase activity, preserving IκBα stability, and preventing nuclear translocation of the NF κB p65 subunit [27]. This action occurs downstream of receptor engagement but upstream of transcriptional activation, resulting in broad suppression of inflammatory gene expression rather than selective cytokine blockade. Through this mechanism, genistein attenuates one of the central drivers of persistent inflammation.
Modulation of JAK/STAT and cytokine amplification. Genistein dampens JAK/STAT signaling by reducing JAK phosphorylation and STAT nuclear localization, particularly within IL 6-STAT3-driven inflammatory loops [26]. By interfering at this level, genistein limits the systemic propagation and stabilization of inflammation rather than its initial triggering. This distinguishes its effects from cytokine specific biologics (e.g. IL1ß antibody therapeutics) and aligns it more closely with network oriented anti inflammatory strategies.
Effects on MAPK signaling and stress responses. Experimental models demonstrate that genistein reduces phosphorylation of p38 and JNK, thereby attenuating MAPK dependent inflammatory amplification and AP 1 transcriptional activity [28]. This mechanism is particularly relevant to stress responsive and senescence linked inflammation, suggesting a role for genistein in mitigating pathways associated with inflammation and aging, inflammaging, rather than acute immune activation.
COX-2 Inhibition. Cyclooxygenase-2 (COX-2) is part of a metabolic pathway that synthesizes pro-inflammatory prostaglandins. Genistein inhibits COX-2 which leads to the reduction of inflammation and pain [29, 30].
Antioxidant properties of genistein Oxidative stress, the imbalance between free radical production and antioxidant defenses, can lead to cell and tissue damage. It is thought to play a critical role in cardiovascular health. Through redox normalization, genistein indirectly suppresses NFκB and MAPK activation while reducing DNA damage and senescence signaling [31]. Genistein acts as a direct antioxidant by scavenging and neutralizing free radicals to prevent their cellular damage [31] and by activating the Nrf2 pathway and upregulating antioxidant gene expression [32].
Genistein and the endothelium Vascular endothelium dysfunction is a pathological condition characterized by the disruption of the balance between vasodilation and vasoconstriction. It is considered an early indicator in the development of atherosclerotic CVD. In addition to its anti inflammatory effects, genistein directly targets endothelial dysfunction which is relevant in PLHIV, perhaps to a higher degree in those with long term cannabis exposure. As a CB1 receptor antagonist, genistein can competitively block delta 9 tetrahydrocannabinol (THC) signaling [24], potentially mitigating cannabis associated vascular inflammation and endothelial impairment. Together, these properties position genistein as a biologically plausible and clinically relevant intervention to address the persistent inflammatory and vascular abnormalities underlying CVD risk in PLHIV receiving ART.
Genistein and the microbiome While genistein is known to be pleiotropic, new evidence suggests genistein modifies the intestinal microbiome positively. Many of the biomarkers associated with cardiometabolic health in our assessments and their physiological activity could also be explained by changes in circulating lipopolysaccharide (LPS) and other inflammatory cytokines regulated as a result of intestinal microbiota change potentially caused by genistein, indicating the monitoring the microbiome during genistein treatment necessary. The elevation of LPS concentration and low-grade chronic inflammation has been associated with mechanisms of the change in intestinal permeability and strongly associated with underlying cardiometabolic disease risk and progression [33]. It has been demonstrated that dysbiosis of the gut microbiota can alter the gut permeability, and several factors can contribute to modifications of the gut microbiota, such as intestinal infections by pathogens, the use of antibiotics, or dietary factors leading to the development of metabolic endotoxemia [34]. Akkermansia muciniphila (phylum Verrucomicrobia) is a mucin-degrading bacterium believed to contribute to intestinal health and glucose homeostasis and improved insulin sensitivity due to reduction in the circulating concentration of LPS [35]. Mice treated with genistein show a selective increase in Akkermansia muciniphila, leading to a reduction of metabolic endotoxemia and insulin sensitivity [36-39]. Furthermore, clinical trials in obese subjects show that consumption of genistein over two months (50mg/day) modifies gut microbiota taxonomy, remarkably increasing the Verrucomicrobia phylum[35]. This selective increase modification of the intestinal microbiota was attributed to the improvement in insulin sensitivity in the patients tested and concomitant reduction in metabolic endotoxemia, increase in AMPK phosphorylation, and expression of genes involved in fatty acid oxidation in skeletal muscle. We expect that genistein will alter the gut microbiome by increasing the presence of the Verrucomicrobia phylum. In PLHIV on ART, gut dysbiosis likely due to microbial translocation plays a central role in maintaining systemic inflammation and cardiovascular risk. Genistein has demonstrated the ability to modulate the microbiome, reduce inflammatory metabolite production, and shift gut barrier function toward a healthier phenotype. Monitoring the gut microbiome during genistein intervention will uncover mechanistic pathways, act as a predictive biomarker of efficacy, and guide potential additional therapeutics.
Given these potential multimodal actions, there is a clear rationale to explore the efficacy of genistein in PLHIV on ART through well-structured clinical protocols. Genistein's side effect profile is considered safe, and it has been consumed as a supplement for many centuries. A critical challenge, however, lies in its low bioavailability. To overcome the low bioavailability, we are proposing a phase I clinical trial to explore the safety and efficacy of higher doses of genistein.
C. Safety Considerations. The cumulative evidence from extensive clinical studies supports the clinical safety of Genistein (see Investigator's Brochure). We have been approved for higher doses up to 1500mg per day in heart failure patients (GASPAR: Genistein In Transthyretin Receptor Amyloid Cardiomyopathy, WREM 122758, ReDA:13179, Appendix 1). The safety concerns for PLHIV have been examined and summarized for over-the-counter (OTC) medications (Table 1), prescription medications (Table 2), ART for PLHIV (Table 3) and in the context of cannabis use which is statistically higher in this population.
D. Specific Objectives The systemic inflammation in PLHIV is well established as well as the limitations of therapies, notably the undesirable side effects of current anti-inflammatory medications. This study aims to investigate the efficacy of genistein in countering the systemic inflammation underlying CVD risk and chronic pain in PLHIV, including those whose vascular dysfunction may be exacerbated by cannabis use. The primary objectives are to evaluate genistein's impact on inflammatory and cardiometabolic biomarkers, as well as vascular function and pain level. This study also incorporates two exploratory objectives: an investigation on whether the microbiome is a key modulator of inflammatory markers and the use of induced pluripotent stem cells (iPSCs) derived from participant blood samples to assess cellular mechanisms influenced by genistein. This complementary approach will provide mechanistic insight into genistein's therapeutic potential in PLHIV.
Objective 1: Investigate whether genistein attenuates systemic inflammation associated with CVD in PLHIV.
Objective 2: Investigate whether the vascular dysfunction found in PLHIV is attenuated by genistein.
Objective 3: Investigate whether genistein attenuates pain, chronic or otherwise, in PLHIV.
Objective 4: Investigate whether genistein in PLHIV alters the microbiome to reduce levels of inflammation.
Exploratory Objective: An optional sub-study will incorporate the use of induced pluripotent stem cells (iPSCs) derived from participant blood samples to assess cellular mechanisms influenced by genistein to gain further insight into genistein's therapeutic potential in PLHIV.
Inclusion Criteria:
Exclusion Criteria:
mark.chandy@lhsc.on.ca519-661-2111
Kerry-Ann.Nakrieko@lhsc.on.ca519-661-2111