Streptococcus mutans has been implicated as a primary causative organism of dental caries, one of the most prevalent diseases in humans (Koga et al., 2002). Many studies have demonstrated associations between oral bacteria and systemic diseases, with S. mutans receiving particular attention (Fang et al., 2024, Xiao and Li, 2025). Several reports have implicated this organism in the development of cardiovascular disease (CVD). For instance, S. mutans DNA has been detected in heart valve and aneurysm clinical specimens using real-time polymerase chain reaction (PCR) (Nakano et al., 2007), and S. mutans has been visualized in atheromatous plaque specimens by immunohistochemical staining (Fernandes Forte et al., 2022). Furthermore, S. mutans has been identified in blood samples from patients with infective endocarditis (Faraji et al., 2018, Kim et al., 2018), and the prevalence of infective endocarditis caused by S. mutans was found to be highest among cases of streptococcal bloodstream infections (Chamat-Hedemand et al., 2020). Epidemiological studies have also revealed that S. mutans strains expressing Cnm protein are associated with an increased prevalence of cerebral microhemorrhages (Watanabe et al., 2016, Hosoki et al., 2020). Animal studies have further demonstrated the pathogenic potential of S. mutans, including the induction of infective endocarditis (Nagata et al., 2006) and acceleration of atherosclerosis (Kesavalu et al., 2012). Previous investigations have shown that S. mutans is capable of invading human aortic endothelial cells (HAECs) and human coronary artery endothelial cells (Nagata et al., 2011, Abranches et al., 2011). Our group has reported that HAECs invaded by S. mutans strains produce higher levels of CVD-related proinflammatory cytokines compared to non-invaded HAECs (Nagata et al., 2011). We have also demonstrated that the cytokine production induced by invasive S. mutans is associated with the upregulation of intracellular toll-like receptor 2 and nucleotide-binding oligomerization domain 2 in HAECs (Nagata & Oho, 2017).

Substance P (SP) is an endogenous neuropeptide belonging to the tachykinin family. SP was discovered as a mediator in nociception and neurogenic inflammation, and its expanded roles in the immune response, inflammation, and homeostasis in non-neural tissues have attracted considerable attention in recent years (Mashaghi et al., 2016, Suvas, 2017). In particular, the involvement of SP in physiological and pathological processes within the cardiovascular system is currently the foci of research (Feickert & Burckhardt, 2019). There have been many studies reporting the molecular mechanisms of SP in the cardiovascular system and CVD (Mistrova et al., 2016, Hoover et al., 2000). Inflammatory processes are involved in the pathogenesis of CVD (Alfaddagh et al., 2020), and one of the causes that trigger inflammation in the cardiovascular system is bacterial infection (Leishman et al., 2010). However, the roles of SP in the interaction between bacteria and cardiovascular tissues have not been fully examined. The potential role of SP in modulating the HAECs response to S. mutans in the context of CVD pathogenesis has not been investigated. SP is expressed in vascular endothelial cells (Linnik and Moskowitz, 1989, Milner et al., 2004). Since endothelial cells are the innermost lining of blood vessels, they are frequently exposed to pathogens and bacterial products that enter the bloodstream. Thus, it is interesting to investigate SP expression in HAECs following S. mutans stimulation and to elucidate its interaction with S. mutans in the context of CVD pathogenesis.

Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin (Ericson & Rundegren, 1983) or gp-340 (Holmskov et al., 1997), belongs to the scavenger receptor cysteine-rich superfamily and plays a critical role in innate immunity, inflammation, and epithelial homeostasis (Reichhardt et al., 2017). As an innate immune receptor, DMBT1 protects mucosal surfaces by binding to and agglutinating pathogens. In a previous study, we demonstrated that DMBT1 is expressed in HAECs following stimulation with S. mutans, and that DMBT1 inhibits S. mutans adherence to HAECs through bacterial agglutination, thereby suppressing both S. mutans invasion and S. mutans-induced cytokine production in HAECs (Oho & Nagata, 2019).

This study investigated the expression of SP in HAECs stimulated with S. mutans and explored the role of SP in the interaction between S. mutans and HAECs, considering its cooperative action with DMBT1. Specifically, we confirmed SP expression and examined its effects on S. mutans invasion of HAECs, cytokine production in S. mutans-stimulated HAECs, and S. mutans-mediated cytotoxicity to HAECs. Our results suggest that SP acts cooperatively with DMBT1 to play an inhibitory role in the pathogenesis of CVD induced by S. mutans.