Atherosclerosis is a chronic inflammatory disease triggered by the accumulation of cholesterol-containing low-density lipoprotein (LDL) particles in the vessel walls (1). An atherosclerosis epidemic has swept across the world, setting the stage for diverse cardiovascular diseases, such as myocardial infarction, angina and stroke (2,3), now accounting for the majority of morbidity and mortality worldwide. Atherosclerosis, which occurs principally in large- and medium-sized arteries and consists of a lipid core and an outer fibrous cap (4), is a complex but coordinated pathophysiological process that involves endothelial damage, macrophage phagocytosis of lipids into foam cells and inflammatory cell infiltration. Investigating the findings of atherosclerosis can promote a better understanding of its pathogenesis. However, the etiology of atherosclerosis remains unclear and difficult to decipher. Thus, the characterization of atherosclerosis is crucial for a thorough understanding.

Recently, the discovery of an attractive group of mediators known as the homeobox (HOX) has revealed causality in various biological processes and diseases. Overwhelming data on HOX genes have highlighted their roles in diverse cancers, such as colorectal cancer (5), breast cancer (6) and lung cancer (7). Burgeoning observations indicate that interrogation of the HOX gene is an emerging area of focus in cardiovascular biology and disease. Previous studies have underscored the role of HOX in the modulation of cardiac and vascular development (8-10). A recent study suggested that HOXA1 participates in the modulation of viability and migration of long noncoding (lnc)RNA ROR-mediated biological characteristics in ox-LDL-induced human umbilical vein endothelial cells (HUVECs) (11). Another study showed that HOXA5 protects against carotid atherosclerosis development by suppressing the phenotypic transformation of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic form via activation of peroxisome proliferator-activated receptor γ (PPARγ) (12). Therefore, these data implicate HOX in cardiovascular biological processes, suggesting that it may be linked to cardiovascular disease. A strong focus has been placed not only on the exploration of their biological function, but also on unscrambling how the HOX gene comes into play at the molecular level in atherosclerosis.

In the present opinion article, the various mechanisms by which HOX gene surveillance influences atherosclerosis and other cardiovascular diseases was primarily highlighted, initially providing a detailed description of HOX gene-mediated pathophysiological processes regarding the onset and progression of atherosclerosis. This is followed by HOX gene involvement revelation in other pivotal biological processes, including cardiac/vascular development, cardiomyocyte pyroptosis/apoptosis, cardiac fibroblast proliferation and cardiac hypertrophy, which are pertinent to diverse cardiovascular problems. Finally, knowledge gaps, intriguing outlook and future directions of HOX in atherosclerosis are discussed. Such findings regarding the HOX gene will provide novel and unexpected insight with major potential to advance our understanding of normal physiology and cardiovascular diseases, including atherosclerosis. Collectively, the present study offers a compendium for understanding new potential targets of atherosclerosis with significance in the amelioration of atherosclerotic cardiovascular diseases. Despite the significant progress thus far, major challenges and conundrums remain.

The HOX gene family is an ancient and highly conserved group of transcription factors (TFs) that modulates master developmental processes, such as anteroposterior body axis patterning, organ morphogenesis and cell fate determination. In humans, 39 HOX genes are arranged in four clusters (HOXA, HOXB, HOXC and HOXD), located on diverse chromosomes (7p15.2, 17q21.32, 12q13.13 and 2q31.1) (13). Each cluster includes 9 to 11 paralog HOX genes, numbered 1 to 13 based on their 3′ to 5′ chromosomal position (Fig. 1A) (14). Initially, HOX was found to be a set of transcriptional modulators that have a crucial role in embryogenesis and body segmentation in Drosophila (15). Structurally, HOX contains two exons and an intron, and its functions depend on an evolutionarily conserved 60 amino acid homeodomain (HD) and a hexapeptide motif (HX) (Fig. 1B). The HD is mainly responsible for DNA binding at specifically recognized sites, leading to transcriptional inhibition or activation of target genes (16,17). HX is indispensable for binding to co-factors, such as members of the three-amino acid loop extension-TALE group (63-amino acid HD) of potential barrier chromatography proteins (18). DNA-binding is determined by helix 3 along the N-terminal arm, which identifies only four base-pair sequences (TAAT, ATTA, TTAT and ATAA), indicating low functional specificity (19). HOX genes located at the 3′ end of the cluster are expressed earlier in development and in more anterior body regions, whereas those at the 5′ end are expressed during later development (20). Functionally, HOX has a pivotal role in embryonic development and multiple adult tissues. HOX is initially expressed during embryogenesis, where it orchestrates a plethora of developmental processes, including limb formation, anterior-posterior axis patterning, craniofacial morphogenesis and the development of the central nervous system. It also modulates a multitude of biological processes, signaling molecules, components of various signaling pathways and other TFs (21). At the cellular level, HOX is a major regulator of various biological processes, including cell death, proliferation, differentiation, migration and apoptosis (17). Mechanistically, the mode of action is mainly via transcriptional activation or repression of target genes (Fig. 1C) (22). In addition, HOX TFs can bind enhancers, promoters and intronic and intergenic regions via interactions with histone-modifying groups and co-factors (23). However, the molecular mechanisms underlying transcriptional regulation by HOX remain unclear and only a few HOX-dependent molecular pathways have been characterized. Hence, substantial efforts are required to elucidate the underlying molecular mechanisms. In addition to its transcriptional regulatory roles, HOX also has non-transcriptional functions. They have roles in DNA damage repair (24), replication (25), translation initiation (26), protein degradation (27) and mRNA processing (26). Therefore, HOX modulates gene expression at multiple levels. The upstream regulatory mechanism of HOX is tightly regulated by a multilayered regulatory system comprising promoter DNA methylation, histone methylation, regulation by other transcription factors and post-transcriptional modulation by non-coding RNA (14).

Probing the molecular and cellular events that occur during atherosclerosis may provide new therapeutic strategies for its development. Recently, there has been increasing interest in unscrambling the HOX gene in cardiovascular diseases (28,29). Atherosclerosis is the most common underlying pathology of cardiovascular disease. Hence, extensive studies have shown that the HOX gene has a central role in atherosclerosis; for instance, HOXA1 participates in atherosclerosis via the microRNA (miR)-99a-5p-HOXA1 axis (30). The development of targeted therapies against HOX in atherosclerosis is a promising research direction. The initiation of atherosclerosis is largely a response to an imbalance in cardiovascular biology. In the following section, the prominent molecular events governed by HOX in atherosclerosis pathogenesis are emphasized, with a particular focus on lipid metabolism, inflammatory response, angiogenesis, cellular proliferation and apoptosis, vascular remodeling and macrophage polarization (Fig. 2 and Table I).

Besides the HOX gene surveillance mechanisms summarized above, other HOX gene surveillance machinery types exist. In the following sections, the functional roles and molecular mechanisms of HOX in cardiac/vascular development, cardiomyocyte pyroptosis/apoptosis, cardiac fibroblast proliferation and cardiac hypertrophy related to cardiovascular problems were discussed (Fig. 3 and Table II).

The identification of non-coding RNAs as functional players has revolutionized the field of RNA biology. In addition to encoding transcription factors, transcription of HOX genes yields non-protein-coding genes, including lncRNAs (93). A broad array of studies has indicated that HOX cluster-embedded lncRNAs (HOX-lncRNAs) are crucial for diverse cancer biology (94,95). In humans, there are 18 antisense RNA genes within the four HOX gene clusters in the National Center for Biotechnology GenBank database (https://www.ncbi.nlm.nih.gov/). HOX-lncRNAs play a major role in modulating the expression of HOX and non-HOX genes (96). Investigations have indicated that HOX-lncRNAs could regulate HOX expression in a cisor trans-regulatory manner. For instance, HOTAIR, a HOX-lncRNA resulting from the HOXC cluster, acts in a trans-regulatory manner and modulates HOXD cluster expression by recruiting polycomb repressive complex 2 in adult human primary fibroblasts (97). HOTAIRM1 can regulate the expression of both 3′ HOXA genes in cis and other genes involved in the alteration of β2-integrin signaling in a trans-regulatory manner (98,99).

Additional HOX-lncRNAs require further investigation and the pivotal effects of HOX-lncRNAs on HOX expression remain unclear. Further, little is known about the mechanisms by which HOX genes govern HOX-lncRNA expression. Hence, further studies are needed to explore these regulatory circuits. HOX-lncRNAs are the most widely studied within the HOX network. HOTAIR is involved in various pathological conditions. Several recent studies have suggested that HOX-lncRNAs have an important role in cardiovascular diseases. For instance, HOTAIR can affect atherogenesis by regulating diverse biological processes, including the inflammatory response (100,101), angiogenesis (102), and cellular proliferation and apoptosis (103,104). These studies have filled a substantial gap in the mechanisms orchestrating cardiovascular disease in HOX-mediated regulatory circuits. An in-depth investigation of HOX-lncRNAs will likely yield novel insight into biological mechanisms and is a promising area for future research on cardiovascular disease. Understanding the roles of HOX genes and how they coordinate their relationship with HOX-lncRNAs will provide a more comprehensive understanding of the relevant cells and biology. However, HOX genes associated with diverse HOX-lncRNAs are yet to be fully functionally and mechanistically characterized. Capitalizing on the signaling pathways previously identified in cancer disorders and deciphering their mechanisms of action are likely to be useful strategies in the cardiovascular field. In addition, miRNAs can regulate the expression of HOX and thus participate in the occurrence and development of atherosclerosis. For example, mi-99a-5p alleviates atherosclerosis via regulating HOXA1 (30). In addition, the miR-17-5p/HOXB13 axis participates in the phenotypic modulation of VSMCs (67). In short, ncRNAs may regulate the expression of HOX genes and have a potential role in atherosclerosis.

Given that numerous studies have implicated HOX genes in regulating diverse cardiovascular biology, there may be opportunities to treat relevant diseases by modulating HOX genes. Several HOX genes, including HOXA5, HOXB5, HOXC6, HOXC8 and HOXB7, are differentially expressed in epicardial adipose tissue and subcutaneous adipose tissue of patients with coronary artery disease (105). Another study showed that HOXB4 serves as a potential diagnostic marker of acute myocardial infarction (106). HOXC5, a hub gene, is differentially expressed in abnormal coronary endothelial function samples compared to their counterparts, suggesting that it may be involved in the development of early coronary atherosclerosis (107). HOXA5 is upregulated in the lungs of patients with pheophytinase compared to normal lung tissue (108). Hence, HOX genes are expected to be useful biomarkers for diagnosing cardiovascular disease. HOX genes structurally occupy rich CPG islands in their promoters. Therefore, methylated HOX genes and HOX-associated histones have been recognized as potential biomarkers in numerous cancers. Epigenetic therapy using histone demethylases and deacetylases inhibitors may represent a promising treatment strategy. However, ample technical and theoretical details must be considered when designing biomarkers to determine their specificity and sensitivity. Precise quantification and determination of value normalization are critical for decreasing variability and are therefore essential for rigorous biomarker interrogation. Further investigations are warranted to identify HOX as a novel class of molecules for therapeutic intervention against cardiovascular disorders. As mentioned above, HOX influences numerous cardiovascular biological processes, particularly atherosclerosis development and progression, by coordinating various pathophysiological processes. Consequently, an in-depth investigation of HOX genes is expected to enable their use as potential diagnostic biomarkers for specific diseases.

Over the past decade, interest in elucidating cardiovascular disease has increased owing to its high morbidity and mortality. In recent years, dysregulation of HOX gene regulatory circuits has been linked to atherosclerosis. Therefore, understanding how various intricate molecular landscapes contribute to atherosclerosis is pivotal. To this end, atherosclerosis-associated investigations have been conducted to elucidate the underlying molecular mechanisms. To a certain extent, the advancement of gene function has led to remarkable alterations in the appreciation of HOX beyond its existing function in bodily development. The present review outlined the state-of-the-art HOX gene for atherosclerosis. Other critical biological processes dominated by the HOX gene, which is essential for cardiovascular diseases, including cardiac/vascular development, cardiomyocyte pyroptosis/apoptosis, cardiac fibroblast proliferation and cardiac hypertrophy, were also discussed. Overall, a preponderance of data supports the notion that HOX dysregulation may be causally linked to several cardiovascular diseases, including atherosclerosis. Thus, we are optimistic that the ongoing investigation of HOX holds outstanding promise to provide new directions with great potential to ameliorate the current bottleneck in treating atherosclerosis. Most findings have yet to be pinpointed in detail. Of the 39 HOX genes in humans, only a few have been exhaustively substantiated, and perhaps even a limited number have a biologically significant impingement on atherosclerosis, let alone translate into the clinic. Taken together, HOX, a previously underappreciated central modulator, is being considered for atherosclerosis, thus showcasing a fascinating yet challenging landscape for developing new therapeutic strategies for atherosclerosis. However, much remains to be done in future journeys.

Mounting evidence has shown HOX involvement in atherosclerosis; however, several core issues concerning HOX go beyond the scope of the aspects presented above. Besides celebrating existing advances, delving into the molecular basis of HOX in atherosclerosis, such as overwhelming molecular insight in vitro and rigorous functional roles in vivo, requires further investigation. Monumental challenges exist in moving forward and substantial knowledge gaps must be addressed in detail.

In the laboratory, a large collection of studies is warranted to elucidate the fundamental far-reaching roles of HOX in atherosclerosis. However, several outstanding questions remain to be resolved: i) The epigenetic mechanisms by which HOX gene expression is crucial for the understanding of diverse cardiovascular diseases are still poorly characterized. An in-depth understanding of the HOX gene epigenetic landscape that dissects pathogenesis during the atherosclerosis process will be needed to provide causality at the transcriptional level. ii) How the HOX gene orchestrates its downstream target genes and how they are targeted to specific genetic loci remain less well documented. iii) The molecular crosstalk between different HOX genes may delineate a subtle network to regulate sequential expression during development (109,110). However, details of the interplay and molecular crosstalk between HOX gene members, as well as key insights in atherosclerosis-harnessing animal models particularly, are still not widely imprinted. iv) As previously mentioned, a large fraction of HOX cluster-embedded lncRNAs have not yet been functionally characterized, particularly in the cardiovascular field. More specifically, the interaction between HOX-lncRNAs and HOX genes remains largely vague; thus, plentiful work underscoring their regulatory layers is necessary to open a new chapter for dissecting the interplay between HOX-lncRNAs and HOX genes in atherosclerosis.

In translational undertakings, perhaps the most challenging aspect is how the scientific discoveries of the HOX gene described herein yield benefits for cardiovascular diseases. Therefore, detailed studies in animal models and rigorous pre-clinical research trials are likely to be paramount in filling this gap. Follow-up work is needed to address whether the changes in HOX gene expression are i) a cause or contributor to atherosclerosis or ii) the effect of atherosclerosis. If HOX gene expression is a cause of atherosclerosis, it may be favorable to develop anti-atherosclerosis therapies that target HOX gene expression. Furthermore, if this is the case, such expression may be helpful as a robust biomarker of atherosclerosis or for evaluating the effect of anti-atherosclerosis therapy. Certainly, to target particular HOX genes in atherosclerosis, ongoing work is imperative to determine causal links between diverse signaling pathways concerning the HOX gene at both the transcriptomic and proteomic levels before clinical application.

In general, the governance of major regulatory mechanisms involving multiple stepwise factors related to atherosclerosis is complex and often requires the integration of multiple layers of control. Consequently, it is impossible to shed light on atherosclerosis onset and progression using a single mechanism, and future efforts to disentangle its pathogenesis and exploit new effective therapeutics will need comprehensive approaches as a prerequisite, taking into consideration all kinds of regulatory mechanisms. A focus addressing these questions is needed to integrate multi-dimensional data derived from cellular, organoid-based, in vivo, molecular, genetic, epigenetic, transcriptomic and proteomic mechanisms, coupled with state-of-the-art technologies that allow detection of more HOX gene characteristics at the level of single cells, leveraging atherosclerosis-relevant tissues and ultimately translate unparalleled molecular findings into clinical practice to benefit patients with atherosclerosis, from the lab to the clinic.