Coronary artery disease (CAD) is a cardiovascular disease (CVD), manifested by stable angina, unstable angina, myocardial infarction, or sudden cardiac death, and is one of the primary causes of death worldwide [1]. Despite advances in diagnostic and treatment technologies in recent years, the prevalence of CAD continues to increase annually, and represents a serious threat to public health [2]. Vascular calcification, especially coronary artery calcification (CAC), is prevalent, harmful, and progresses rapidly in patients with CAD. Previous studies have shown that the presence of CAC increases the risk of coronary heart disease events by threefold [3]. Pathological studies have demonstrated a strong correlation in the initiation and progression between CAC and CAD [4]. CAC is often located in areas of atherosclerotic lesions [5]. The severity of CAC and the degree of coronary stenosis directly impact the management and treatment of CAD. In addition, it is difficult to perform treatments to eradicate CAC. Thus, early anticipation of the high risk of CAC and timely intervention are pivotal for the treatment and prognosis of patients with CAD.

The conventional risk factors, such as race, advanced age, male gender, smoking, diabetes mellitus (DM), hypertension, hyperlipidemia, and chronic kidney disease (CKD), associated with the presence and development of CAC have been widely recognized in the general population [3, 6, 7, 8]. In previous views, the formation of CAC was believed to be caused by the ectopic deposition of calcium salts in the walls of coronary vessels, which was considered as a passive and degenerative pathological phenomenon. However, recent studies support a concept that CAC is an active and regulated process in atherosclerosis progression, reflecting a broader systemic inflammatory response [5, 6]. A study demonstrated that as atherosclerosis progresses, inflammation aids in the initiation and progression of calcification as macrophages secrete inflammatory cytokines and promote osteogenic differentiation of vascular cells [9]. Inflammasomes derived from macrophages, can be activated by various cardiovascular risk factors and drive downstream signaling events. Studies have shown that the inflammasomes nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1) and NLRP3 are closely associated with CVD [10, 11]. Therefore, the inflammasomes NLRP1 and NLRP3 may be able to potentially detect high-risk populations and improving the ability to predict the occurrence of CAC.

The aim of this study was to determine whether the inflammasomes NLRP1 and NLRP3 could serve as a “risk integrator” for CAC, adding predictive information beyond conventional cardiovascular risk factors. Since there is a paucity of research in this area, we conducted a clinical data analysis to assess the relationship between NLRP1, NLRP3 and CAC. We developed a simple and cost-effective nomogram-illustrated model aiming to predict the occurrence of CAC in patients with CAD and to improve therapeutic decisions leading to the primary prevention of CAC.

Two key findings were identified in this study. First, the NLRP1 inflammasome was found to be an independent predictor of CAC. Second, we proposed and validated a valuable prediction model, embracing an extensive set of clinical risk factors that are easily accessible, such as age, smoking, DM, hyperlipidemia, ALP and TG, and incorporating the serum NLRP1 level. This web-based dynamic nomogram model (https://jingfengpeng.shinyapps.io/DynNomapp/) can soon be obtained for free online. The results generated from this model could serve as a guide for preventing or even slowing down the progression of CAC.

CAC plays an important role in CAD. The majority of mortality and major adverse events in cardiovascular disease are related to CAD, in which CAC plays a significant role. A significant correlation was found between the presence and extent of CAC and the overall magnitude of coronary atherosclerotic plaque burden, as well as the development of subsequent coronary events [3, 9]. Coronary calcification may deteriorate vascular compliance. Calcified plaques demonstrating a spotty pattern in coronary arteries are considered to increase the risk of plaque rupture [12, 13]. The progression of CAC, not only contributes to the risk of cardiovascular mortality, but also increases the difficulty for intervention therapy. CAC has always been a significant challenge for interventional cardiologists. Efforts to control CAC with medical therapy have not been successful. While advances in percutaneous techniques have modestly improved the outcomes of percutaneous coronary intervention (PCI), the risks and adverse events associated with the treatment of recalcitrant calcified lesions remains high. In addition, the treatment of CAC increases medical costs [6]. Therefore, it is necessary to expand therapies beyond mechanical revascularization to encompass predictive diagnosis and preventive interventions to treat CAC.

In patents with CAC, inflammation has been underestimated in previous prediction models. In recent years, emerging research suggest that the initiation and progression of CAC are collaboratively driven by long-term dyslipidemia and vascular inflammation, which are the basis of atherosclerosis [5, 9]. A study found that the greater extent of CAC among patients with severe rheumatoid arthritis was due to the effect of inflammatory mediators, which confirmed the strong impact of inflammation in the pathogenesis of CAC [14]. A major participant in the inflammatory response of CAC are macrophages, which further promote disease progression in a positive-feedback amplification loop of calcification and inflammation [9]. Proinflammatory stimuli induced by CVD promote the majority of inflammasome specks to accumulate in granulocytes and macrophages during the progression stage of inflammation [15, 16, 17]. We developed a strong interest in inflammasomes from earlier studies reports about inflammasomes and their derivation from macrophages [18, 19]. Several studies have found that there were numerous pattern-recognition receptors (PRRs) capable of assembling the inflammasome complex, but the well-established inflammasomes were still NLRP1, NLRP3, nucleotide-binding domain (NOD)-like receptor family caspase-associated recruitment domain-containing protein 4 (NLRC4) and absent in melanoma 2 (AIM2), among which NLRP1 and NLRP3 were the most widely studied in CVD [20]. Accumulating evidence supports that inflammasomes, capable of triggering and modulating inflammation-related signaling pathways, play the crucial role in the progression of various CVD [10, 21, 22, 23]. Studies have reported that the protein expressions of NLRP3 and caspase-1 in circulating monocytes among patients with acute coronary syndrome were increased [22]. Similarly, the NLRP1 inflammasome were found to increase in patients with primary atherosclerotic lesions and inflammasome complex was activated by interaction with NLRP1 and NLRC4 receptors [24]. In the progression of atherosclerotic lesions, cholesterol crystals were found to directly activate the NLRP3 inflammasome [25]. Elevated levels of triglycerides and very-low-density lipoprotein cholesterol stimulated activation of the NLRP1 inflammasome by nuclear factor kappa-B (NF-$\kappa$B) [24, 26]. Additionally, interleukin-1$\beta$ (IL-1$\beta$) and IL-18, downstream proinflammatory cytokines of inflammasomes, were also found to affect the development and stability of atherosclerotic plaques [27, 28]. These influencing factors subsequently combine lipid metabolism and inflammation to exacerbate disease progression. Therefore, we investigated whether NLRP1 or NLRP3 might be related to CAC. We found that a higher serum level of NLRP1 resulted in an increased CAC risk in patients with CAD. The NLRP3 inflammasome, the most widely explored inflammasome, was found to be unrelated to the prevalence of CAC. Further studies are needed to explain these novel findings.

Advanced age is a well-established risk and prognostic factor for CAC. Our logistic analysis revealed that patients with advanced age had a significantly increased risk of CAC, consistent with previous studies [3, 7]. Cigarette smoking, a significant health, remains highly prevalent worldwide and contributes to cardiovascular morbidity and mortality. Our findings, consistent with prior studies, showed that cigarette smoking was a critical factor of the presence and extent of CAC [29, 30]. Nicotine in cigarettes increases secretion of inflammatory cytokines and elevates lipid content within atherosclerotic lesions, subsequently causing osteogenic differentiation of vascular smooth muscle cells (VSMCs) [31]. CAC has also been found to be more severe in patients with DM [3, 8]. Our findings parallel prior studies on the relationship of DM and CAC. The mechanism of CAC induced by DM can be attributed to multiple factors. The main metabolites of diabetic individuals, advanced glycosylation end products (AGEs) can contribute to oxidative stress and the inflammatory response. Long-term exposure of VSMCs to a high glucose environment can activate relevant signaling pathways such as Wnt, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-$\kappa$B, and increase the expression of Runt-related transcription factor 2 (Runx2) and Osterix (Osx), which are the key transcription factors that accentuates osteoblast‑like differentiation of VSMCs [5, 7, 32, 33]. In this study, we also found that a history of hyperlipidemia was significantly correlated with an increased risk for CAC. Experimental and clinical data have shown that hyperlipidemia not only promoted atherosclerotic plaque development, but also increased vascular calcification [4, 12]. The levels of oxidized phospholipids (ox-PLs) and oxidized low-density lipoprotein (ox-LDL) are elevated in the serum of patients with hyperlipidemia, which increases oxidative stress and the inflammatory response in the endothelium, as well enhancing the phenotypic transition of VSMCs into mature osteoblasts and mineralization by upregulating Osx expression, thereby leading to the initiation of CAC [34, 35]. Similar to our results, several animal experiments and clinical trials have confirmed a role of TG in the prevalence of CAC [35, 36]. Decreases TG levels can reduce the CAC Agatston score, a scoring method to estimate and quantify the extent of CAC [37, 38, 39]. This study also found that the serum ALP level was an independent risk factor of CAC. Several research studies have demonstrated that ALP, a key enzyme in vascular calcification, can hydrolyze phosphate bonds, inducing local accumulation of phosphate, which provides a microenvironment for calcification. Moreover, the activated ALP by various stimuli can provoke or modulate the osteoblast‑like differentiation of VSMCs [40, 41, 42].

There are several limitations in this study. First, patients with certain diseases such as severe CKD, were more likely to prioritize receiving treatment from other departments, rather than undergoing CAG directly because of concern for increased complications. Lack of the data from these patients might affect the robustness of the model. Second, to prevent bias of insufficient data, some previously reported risk factors of CAC, such as the serum levels of parathyroid hormone (PTH), vitamin D, calcium and phosphorus, were not included. Third, this is a single-center study focused on the Chinese population. More information, such as ethnic background, diet and physical activity, awaits clarification in future studies to make the model more compatible and generalizable. In addition, to ensure the credibility and robustness of our model, more external data are warranted for validation in future studies.

This study found that the serum NLRP1 level was an independent risk factor of CAC in patients with CAD. We developed a web-based dynamic nomogram model consisting of 7 clinical characteristics, which may serve as a simple-to-use screening tool to personalize the risk of developing CAC and improve the therapeutic options for patients with CAD.

All data that support the findings in this study are not publicly available due to patient privacy, but are available from the corresponding author upon reasonable request.

JFP, BHZ, WHL, WHQ, JZ and FFL made substantial contributions to conception and design of the work, analysis and interpretation of data, took part in drafting, revising and critically reviewing the manuscript; TX, XBH, YHZ, YXW and SYP contributed in acquisition of data, analysis and interpretation of data, been involved in revising and critically reviewing the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (No: XYFYLW2017-002). All patients understood the study procedure and voluntarily signed an informed consent form.

Not applicable.

This research was supported by the National Natural Science Foundation of China (Grant No.81900216) and the Science and Technology Program of Xuzhou (KC21067).

The authors declare no conflict of interest.