Coronary artery disease (CAD) stands as one of the foremost contributors to cardiovascular events and death on a global scale [1]. The primary underlying mechanism for CAD is atherosclerosis, which can precipitate severe cardiovascular events, including myocardial infarction and stroke. Despite significant advancements in clinical diagnosis and treatment modalities, the incidence and mortality rates associated with CAD remain alarmingly high, necessitating continuous research into novel biomarkers and therapeutic targets.

Recent investigations have illuminated the importance of circulating non-coding RNAs (ncRNAs) as promising biomarkers in the landscape of cardiovascular diseases (CVDs) [2-4]. Among these, long non-coding RNAs (lncRNAs) have garnered attention for their multifaceted roles in cellular regulation and their potential as therapeutic targets. Notably, extracellular vesicles (EVs) have emerged as significant players in the pathophysiology of various CVDs, serving as carriers for these regulatory molecules. The ability of EVs to transport lncRNAs presents an intriguing avenue for understanding the molecular underpinnings of CAD.

A pivotal study conducted by Hosen et al. [5] has made strides in elucidating the role of the lncRNA PUNISHER in CAD. This research addresses a critical gap in our understanding of how EVs facilitate the intercellular transport of lncRNAs and their subsequent impact on endothelial cell (EC) function and angiogenesis. The study underscores the importance of small extracellular vesicles (sEVs) in this context, showcasing how they can mediate the regulatory functions of PUNISHER in the vascular system. Interestingly, the findings of this study reveal a novel mechanism wherein lncRNA PUNISHER promotes the pathophysiology of CAD by interacting with the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNPK). This interaction is crucial for the regulation of vascular endothelial growth factor A (VEGFA), a proangiogenic protein that plays a vital role in endothelial function and angiogenesis. By elucidating this pathway, the study significantly enhances our understanding of the molecular mechanisms that drive CAD. Additionally, the exploration of lncRNA PUNISHER within the context of EVs sheds light on the regulatory networks underlying endothelial biology and opens up promising avenues for therapeutic intervention. By targeting the interactions between lncRNAs, proteins, and EVs, there lies the possibility of developing innovative strategies to mitigate the progression of CAD. Ultimately, the investigation into circulating non-coding RNAs and their transport via extracellular vesicles is an exciting frontier in cardiovascular research. The work by Hosen et al. marks a significant contribution to our understanding of CAD, particularly in the context of lncRNA PUNISHER and its regulatory roles. As we continue to uncover the complex molecular landscape of CAD, such insights will be invaluable in the pursuit of effective treatments and improved patient outcomes.

Over the past few decades, the intricate molecular mechanisms contributing to the development and progression of CAD have garnered significant interest within the scientific community. Among the promising areas of research is the role of lncRNAs encapsulated in EVs. This exploration sheds light on the complexities of CAD pathology while simultaneously paving the way for innovative diagnostic and therapeutic strategies [7].

Extracellular vesicles, which include small EVs (formerly known as exosomes) and large EVs (formerly known as microvesicles), are lipid-bilayered particles secreted by nearly all cell types. These vesicles are crucial mediators of intercellular communication, facilitating the transfer of various bioactive molecules, including proteins, lipids, mRNAs, microRNAs (miRNAs), and lncRNAs-between cells. Their ability to foster crosstalk between diverse cell types, such as endothelial cells, smooth muscle cells, and immune cells, is particularly significant in the context of CAD. These interactions are integral to processes such as inflammation, endothelial dysfunction, and vascular remodeling, all of which are critical components of CAD pathogenesis.

Historically viewed as “junk” RNA, lncRNAs have recently emerged as vital regulators of numerous cellular processes relevant to CAD. They influence critical pathways, including lipid metabolism, inflammatory responses, and apoptosis, by modulating gene expression at multiple levels—transcriptional, post-transcriptional, and through epigenetic modifications [8]. The encapsulation of lncRNAs within sEVs enables their transfer between cells, allowing them to potentially alter recipient cell function and thus contribute to the progression of CAD [1].

Research has demonstrated that specific lncRNAs packaged within sEVs can exacerbate endothelial dysfunction, a hallmark of early atherosclerosis. This dysfunction sets the stage for a cascade of pathological events, including increased vascular permeability and inflammation, further propelling the development of CAD. Furthermore, certain lncRNAs have been implicated in the modulation of smooth muscle cell proliferation and migration, processes that contribute to plaque instability and the risk of thrombosis, ultimately leading to acute coronary syndromes. Given the emerging evidence linking lncRNAs encapsulated in sEVs to disease mechanisms, there is a compelling case for their potential as novel diagnostic biomarkers. Early detection and prognostic assessment are critical in managing CAD, and lncRNAs may serve as valuable indicators of disease progression. Moreover, understanding the specific roles of these lncRNAs in CAD could lead to the development of targeted therapeutic strategies, aimed at mitigating their pathological effects and improving patient outcomes [9,10].

Recent advancements in molecular genetics have elucidated the multifaceted roles of lncRNAs in various pathological conditions [10,11]. In a pioneering study by Hosen et al., the lncRNA PUNISHER (also known as AGAP2-AS1) has been identified as significantly upregulated in patients with CAD. This research, utilizing integrated multiomics-based methodologies, demonstrates the potential of lncRNA PUNISHER as a CAD biomarker and reveals its functional importance in angiogenesis—a fundamental process in cardiovascular health and disease [5].

The study reveals that PUNISHER is particularly enriched in the plasma-derived sEVs of stable CAD patients. This finding suggests a potential link between myocardial ischemia and the upregulation of PUNISHER, which may enhance neovascularization. The research demonstrates that PUNISHER can influence the angiogenic capabilities of recipient ECs through the transport of PUNISHER-integrated sEVs. The implications of such intercellular communication are profound, as they could shift the paradigm of therapeutic approaches aimed at improving vascular function in CAD.

In vitro analyses elucidated the mechanistic underpinnings of PUNISHER's angiogenic activity. The research indicates that the pharmacological inhibition of PUNISHER expression leads to compromised angiogenic responses and decreased EC proliferation. This impairment highlights PUNISHER's essential role in regulating angiogenesis. Furthermore, the study identifies a selective export mechanism mediated by the hnRNPK, a multifunctional RNA-binding protein crucial for packaging PUNISHER into sEVs. Upon uptake by target ECs, PUNISHER facilitates the elevation of VEGFA mRNA and protein levels, which are vital for endothelial proliferation and migration. However, the study also introduces critical considerations regarding the dual role of PUNISHER in CAD. While its role in promoting angiogenesis may offer therapeutic avenues, the participation of PUNISHER in intraplaque angiogenesis, particularly within patients harboring stable plaques raises concerns about the potential for plaque destabilization and rupture. Understanding this duality is paramount for future clinical applications, as it emphasizes the need for a balanced approach when targeting angiogenic pathways in CAD management.

Furthermore, the findings prompt deeper investigations into the mechanistic pathways linking PUNISHER to other pathological settings, such as cancer. The implications of lncRNA PUNISHER in both cardiovascular and oncological contexts reveal a rich landscape of intercellular communication that is yet to be fully explored. The study by Hosen et al. serves as a stepping-stone for future research, emphasizing the necessity for multifaceted investigations that can unravel the complex roles of lncRNAs like PUNISHER in health and disease. In context of clinical view, the identification of PUNISHER as a significant player in CAD underscores its potential as both a biomarker and a therapeutic target. This research deepens our understanding of angiogenesis in cardiovascular diseases and creates new opportunities for therapeutic interventions to improve patient outcomes. In our study, we found that the pro-angiogenic lncRNA PUNISHER is upregulated in the plasma EV of stable CAD patients. In vitro, PUNISHER regulates the antiangiogenic function of recipient EC via EV transportation. In the best-case scenario, the increased level of PUNISHER in EV, possibly due to chronic ischemia, could ameliorate myocardial ischemia in CAD patients by promoting neovascularization, which may be beneficial for patients with myocardial infarction. However, in patients with stable plaques, the participation of PUNISHER in intraplaque angiogenesis and the potential for plaque rupture could also be detrimental. Hence, these findings are important to understanding the mechanism of angiogenesis in CAD as well as other pathological settings (e.g. cancer in which blocking this lncRNA to inhibit angiogenesis may be beneficial to stop tumor growth and metastasis), keeping in mind that future studies are still required to provide deeper explanations of the clinical significance. A continued exploration of the functional roles and regulatory mechanisms of lncRNA PUNISHER will undoubtedly enhance our grasp of its clinical significance, paving the way for innovative strategies in the management of CAD and related pathological conditions (Figure 1).

One of the most promising implications of this research lies in its potential to inform the development of novel diagnostic tools. As the study highlights, liquid biopsies that track circulating lncRNAs like PUNISHER in sEVs could significantly advance early detection and prognosis in CAD patients. Such non-invasive diagnostic approaches could offer critical insights into disease progression and patient management. Early detection is paramount in CAD, where timely intervention can dramatically alter outcomes. By identifying specific lncRNAs that correlate with disease states, clinicians could adopt more targeted and personalized treatment strategies, ultimately enhancing patient care.

Moreover, the exploration of lncRNAs in this context opens up new avenues for therapeutic interventions. Understanding how these molecules interact within the cellular environment could lead to the development of lncRNA-based therapies that ameliorate the effects of CAD. Such innovations could potentially reduce the burden of cardiovascular diseases, which remain a leading cause of morbidity and mortality worldwide.

Taken together, the aforementioned study marks a significant step forward in cardiovascular research by integrating the role of lncRNAs into the broader understanding of CAD. By expanding the scope of investigation beyond miRNAs and emphasizing the functional relevance of lncRNAs in EV-mediated communication, this research not only advances scientific knowledge but also heralds the advent of novel diagnostic and therapeutic strategies. As the field continues to evolve, ongoing investigations into lncRNAs will undoubtedly contribute to more effective management of cardiovascular diseases, paving the way for improved patient outcomes.

The study effectively demonstrates the significant role of PUNISHER in CAD through a combination of in vitro models and patient samples, utilizing advanced methodologies such as RNA sequencing (RNA-seq), RNA immunoprecipitation (RIP) and quantitative reverse transcription polymerase chain reaction (qRT-PCR). These techniques elucidate the molecular interactions between PUNISHER and hnRNPK, revealing insights into its selective packaging into sEVs and its impact on VEGFA expression. Nonetheless, emerging research indicates that PUNISHER may also exhibit anti-apoptotic and anti-mitochondrial fission effects in vascular smooth muscle cells by targeting miR-664a-5p and OPA1, raising questions about the potential role of sEVs in this context [12]. The inconsistent findings from different cohorts, such as the recent 2022 study in Egypt regarding the PUNISHER rs12318065 A/C variant, highlight the complexities of CAD, influenced by multifactorial elements like age and race [13]. To advance our understanding of the disease, it is imperative for future research to broaden patient cohorts and assess the collective effects of various lncRNAs in sEVs, ultimately aiming to validate PUNISHER as a reliable biomarker across different stages of CAD progression.

In CAD, angiogenesis facilitates the growth of atherosclerotic lesions; angiogenesis within plaques plays a crucial role in plaque destabilization and rupture. In our study, we found that the pro-angiogenic lncRNA PUNISHER is upregulated in the plasma sEVs of stable CAD patients. In vitro PUNISHER was found to regulate the angiogenic function of recipient ECs via the transport of PUNISHER-incorporated sEVs. This result indicates that the increased level of PUNISHER in sEVs might be triggered by ischemia and can ameliorate myocardial ischemia in CAD patients by promoting neovascularization.

However, in patients with stable plaques, the participation of PUNISHER in intraplaque angiogenesis and the potential for a resulting plaque rupture should also be considered. Conversely, these findings are important to understand the mechanism of angiogenesis in CAD as well as other pathological settings (cancer), hinting that future investigations are required to provide deeper explanations for the clinical significance.

Recent data indicating that the involvement of lncRNAs in CVD increases the potential for therapeutic manipulation of lncRNA-mRNA-regulated processes [12-14]. However, targeting cardiovascular cells to achieve the expected benefits is still more challenging in comparison to other major organs (kidney or liver), which are yet to be investigated. For instance, intravenous or intraperitoneal administration of antagomiRs (miRNA inhibitors) requires a prolonged single- or repetitive dosing with a high concentration (approximately 0.5-25 mg/kg body weight) to target miRNAs in the heart or vasculature. Due to the heterogeneity of lncRNA expression in different tissue and cell types, it is undeniably challenging to determine an effective dose, which also depends on the target sequence and the chemistry of the lncRNA therapeutics due to less sequence or species conservation (cellular uptake, stability, pharmacodynamics, lncRNA-target genes, bio-distribution, bio-availability, etc.). The current research underscores the promising role of EV-lncRNAs as liquid biopsy candidates, which could enhance the diagnosis and monitoring of atherosclerosis (e.g. CAD) while offering insights into disease propensity through their distinct expression patterns across various biofluids and conditions. Ultimately, these findings illuminate the dual role of sEV-incorporated PUNISHER as both a biomarker and a modulator of vascular integrity, indicating its potential impact on therapeutic strategies.

Since no inflammatory responses or other complications were observed in our experiments with injected EVs in vivo, transfection of suitable donor cells with desired lncRNAs (or a mixture of several lncRNAs) to subsequently isolate EVs from the supernatant of these cells followed by intravenous injection into a disease model will unveil a differential gene expression in recipient cells and mice. The specific deliveries of therapeutic RNAs are still troublesome. EV-therapeutics will be coupled to a cell-specific (e.g. endothelial cells) promoter or aptamer (a single-stranded DNA/RNA molecule with cell specificity) such that they exert their biological activity only in the corresponding target tissue (tissue tropism, e.g. heart). This aptamer-guided EV-RNA delivery is now useful in cancer diagnosis and can be designed to use for cell-specific EV delivery by chemically attaching cellular markers with high specificity.

This strategy via lncRNA could increase, until now, a limited number of options for enhancing beneficial lncRNA-expression levels in vivo. A careful selection of donor cells might enable the selective targeting of tissues or a limited number of target cells (endothelial cells) in vitro based on cell-specific membrane-bound receptors, and limit off-target effects. In contrast to many of the cellular mediators of CVD, in particular, which may be difficult or impossible to target, EV-lncRNAs represent a feasible option to be targeted by drugs or other therapeutic approaches.

In conclusion, the exploration of circulating ncRNAs, particularly EV-incorporated lncRNAs like PUNISHER, marks a promising frontier in cardiovascular research [14,15]. This work not merely deepens our comprehension of CAD but also unlocks thrilling prospects for the utilization of these molecular entities in clinical practice. As we continue to unravel the complexities of ncRNAs and their roles within the cardiovascular system, we stand on the brink of transformative advancements that could significantly enhance diagnostic accuracy and therapeutic efficacy in combating cardiovascular diseases. The future holds the potential for harnessing these insights into practical applications, ultimately improving patient outcomes in the face of a global cardiovascular crisis.