TO THE EDITOR
The recent publication by Wang et al[1] offers valuable insights into the role of phospholipase D2 (PLD2) as a potential biomarker for assessing the severity of acute pancreatitis (AP). Their study highlights the involvement of PLD2 in critical pathophysiological processes, including neutrophil migration, the inflammatory response, apoptosis, cell migration, and adhesion in pancreatic disease. Furthermore, the authors emphasize the validation of PLD2 as a biomarker and point to its promising potential as a future therapeutic target.
Acute and chronic pancreatitis: Current situation and research challenges
Currently, understanding the early progression of AP during the initial hours of hospitalization is essential for identifying patients at higher risk of developing organ failure, thereby facilitating the implementation of timely therapeutic interventions. It is important to note that AP is one of the leading causes of hospitalization in gastroenterology wards and imposes a substantial economic burden on healthcare systems[2]. However, due to the disease’s complexity and the incomplete understanding of its underlying mechanisms, several prognostic scales and biomarkers have been proposed, although none have shown sufficient accuracy. Most of these tools are designed to evaluate prognosis and severity after 48 hours, by which point the inflammatory cascade is already underway, and they rely on numerous parameters that are not routinely measured.
As a result, there has been a growing interest in identifying simpler and more precise biomarkers to better predict the course of this disease. Evidence suggests that decreased levels of PLD2 are associated with increased neutrophil chemotaxis and the release of pro-inflammatory cytokines, which amplify both pancreatic and systemic inflammation. This association was demonstrated in a recent study by Niu et al[3], which reported that PLD2 expression significantly decreases as the severity of AP increases. Specifically, patients with severe AP showed lower PLD2 protein levels compared with those with moderate or mild disease (P < 0.05), and a strong negative correlation was observed between PLD2 expression and disease severity (r = -0.75, P = 0.002). Moreover, the use of a PLD2 inhibitor significantly enhanced neutrophil migration across all severity groups (P < 0.05), supporting the hypothesis that PLD2 acts as a negative regulator of neutrophil chemotaxis. These findings suggest that PLD2 may play a protective role during the early phase of AP by restraining neutrophil infiltration and limiting the ensuing inflammatory response.
Based on these findings, we advocate for the initiation of prospective studies that systematically measure PLD2 during the early stages of AP progression. To enhance the feasibility and reproducibility of such studies, the sample size should be calculated based on preliminary data reflecting expected differences in PLD2 levels between groups. A follow-up period of at least 7 to 14 days is suggested to capture the dynamic changes of PLD2 during the acute inflammatory phase. Control groups should include both healthy individuals and patients with mild AP to discern baseline levels and disease-specific alterations. Such lines of investigation would enable early intervention, continuous monitoring, and appropriate management. Additionally, this approach could facilitate the development of targeted therapies aimed at modulating the inflammatory cascade, thereby preventing both systemic and local complications. In the long term, this strategy could potentially reduce morbidity, mortality, length of hospital stays, and the overall economic burden associated with AP. The compelling data presented here underscore the essential need for continued investigation into PLD2’s role in the pathophysiology of AP. The goal should be to develop novel diagnostic and therapeutic strategies capable of enhancing patient outcomes.
Given that PLD2 appears to play a pivotal role in modulating the inflammatory response, particularly through the regulation of neutrophil migration, a critical question arises: Could PLD2 be considered a viable therapeutic target to prevent recurrent AP, especially in hereditary, alcoholic, or idiopathic forms? This hypothesis outlines a promising research strategy that could guide the development of more effective and personalized interventions in this challenging clinical landscape. In view of the inflammatory regulatory role of PLD2 in AP, its role in chronic pancreatitis is worth exploring. Chronic pancreatitis is characterized by sustained inflammation, parenchymal remodeling, and progressive fibrosis. It is crucial to explore whether the persistent expression of PLD2 contributes to the chronic activation of the immune system and the maintenance of an inflammatory microenvironment, both of which are central to disease progression. Investigating this dimension would, not only deepen our understanding of the underlying mechanisms, but also identify novel therapeutic targets aimed at preventing complications, such as exocrine pancreatic insufficiency.
Furthermore, it is noteworthy that prior research has implicated PLD2 as a key regulator in alcohol-induced liver disease, a condition sharing many pathogenic mechanisms with chronic pancreatitis. Studies have shown that ethanol exposure increases PLD2 expression in hepatocytes, and its inhibition attenuates the transcription of pro-inflammatory and lipogenic genes, reduces steatosis, and mitigates liver damage. This suggests the possibility that a similar mechanism may be operative in alcoholic chronic pancreatitis, which remains the most common etiology of the disease. Exploring this point could provide valuable insights into the potential for targeting PLD2 in both alcoholic liver and pancreatic diseases[4].
Nevertheless, uncertainty persists regarding whether PLD2 actively regulates these inflammatory processes or if its expression is merely a consequence of inflammation as a secondary phenomenon. This ambiguity is heightened by the observation that, while PLD2 levels tend to be reduced in severe AP, they may be elevated in chronic pancreatitis, suggesting a potential role in sustaining the inflammatory microenvironment. A possible explanation for this apparent contradiction may lie in the pathophysiological differences between the acute and chronic phases of the disease, as well as in the underlying etiology and external factors, such as alcohol exposure. In AP, decreased PLD2 levels could reflect an initial negative regulatory mechanism aimed at limiting excessive neutrophil infiltration and systemic inflammation, whereas in chronic pancreatitis, characterized by persistent inflammation and tissue remodeling, PLD2 overexpression may result from sustained immune activation or represent an adaptive response to continuous stimuli. These findings suggest that PLD2 expression and function may be modulated by disease stage, type of injury, and specific etiological factors, and that its role may not follow a linear trajectory. This highlights the need to reconsider its pathophysiological significance within a dynamic and multifactorial framework.
In this regard, it is noteworthy that other phospholipase isoforms have also been implicated in chronic pancreatitis[5,6]. For instance, type II phospholipase A2, produced in acinar and ductal cells, has been reported as being overexpressed in areas with greater histological damage during episodes of AP and acute attacks of recurrent chronic pancreatitis. These enzymes, along with other pro-inflammatory mediators, such as cytokines, growth factors, and digestive enzymes, could initiate a sustained inflammatory cascade in the pancreas, promoting cellular damage and the replacement of functional pancreatic tissue with connective tissue[7].
In this context, PLD2 emerges as a complementary player to PLA2 in the inflammatory response. PLD2 generates phosphatidic acid, a lipid mediator that modulates early intracellular signaling pathways, including inflammasome activation and membrane remodeling. In contrast, PLA2 releases arachidonic acid, a precursor of proinflammatory eicosanoids that amplify inflammation and directly contribute to tissue damage. Importantly, PLD2 activity may facilitate the subsequent activation of PLA2, establishing a synergistic mechanism that intensifies pancreatic inflammation and may accelerate disease progression[1]. These findings highlight the importance of continuing to explore the role of PLD2, not only in the early stages of AP, but also in the progression to chronic pancreatitis.
PLD2 and its role in pancreatic cancer and other diseases
PLD2 has been linked to a variety of diseases, including vascular disorders, immune alterations, and neurological conditions, such as Alzheimer’s disease. Its overexpression has also been implicated in several types of cancer, including bladder[8], breast[9], prostatic[10], ovarian[11] cancers, as well as hepatocellular carcinoma[12] and colorectal cancer[13,14]. Moreover, PLD2 may contribute to cancer progression through an exosome-mediated pathway[15]. Recent studies using murine models have shown that inhibition or genetic deletion of PLD activity significantly reduces tumor growth and metastatic potential. In particular, PLD2 plays a central role in tumor angiogenesis by modulating critical pathways that regulate endothelial cell proliferation, migration, and vascular permeability. One of the main mechanisms by which PLD2 exerts its pro-angiogenic effects involves the vascular endothelial growth factor (VEGF) signaling pathway. Phosphatidic acid, a product of PLD2 enzymatic activity, enhances VEGF expression and promotes activation of VEGF receptor 2 in endothelial cells, thereby facilitating angiogenic sprouting and neovascularization.
As tumors grow, they require expansion of the tumor vasculature to ensure an adequate supply of oxygen and nutrients. However, the tumor microenvironment is often characterized by hypoxia due to abnormal and inefficient vascular networks. This persistent hypoxia leads to the stabilization of the transcription factor hypoxia-inducible factor 1α (HIF-1α), a key regulator of the angiogenic response. Notably, PLD activity has been shown to enhance both the translation and stabilization of HIF-1α, thereby amplifying pro-angiogenic signaling. In turn, HIF-1α upregulates PLD2 expression via VEGF-mediated activation of Src kinase, establishing a feed-forward loop that promotes pathological neovascularization[16].
These findings suggest a potential role of PLD2 in pancreatic cancer. Research into PLD2 could provide valuable insights into disease staging and prognosis, as well as the evaluation of tumor vascularization, which is crucial for chemotherapy effectiveness. Chemotherapy remains a cornerstone in pancreatic cancer treatment, but its efficacy can be limited by poor tumor vascularization, which impedes the proper distribution of therapeutic agents. Future studies are needed to evaluate a clear relationship between PLD2 and vascularization, to evaluate the role of PLD2 in pancreatic cancer.
Validation of PLD2 considering sex differences
Considering the emerging research on this biomarker, it is crucial to address sex distinctions. The study by Wang et al[1] highlights the relevance of PLD2 as a biological signaling molecule but does not explore whether PLD2 levels vary between men and women. Some studies have shown that sex hormones can alter the expression of various enzymes, including phospholipases. Estrogens have demonstrated protective and anti-inflammatory effects in AP models[17]. Testosterone suppresses PLD activity, which affects the formation of 5-lipoxygenase products. According to the study results by Pergola et al[18], PLD activity and diacylglycerol formation were 1.4- to 1.8-fold lower in male monocytes compared with female monocytes, indicating a significant decrease in PLD activity due to the presence of testosterone. This decrease in PLD activity contributes to lower leukotriene production in male monocytes. Although these results refer to total PLD activity rather than PLD2 specifically, they highlight the potential for androgen-mediated regulation of phospholipase activity and support the relevance of investigating sex-specific differences in PLD2 expression and function. Hormonal fluctuations, particularly in women, could affect the expression and function of PLD2. Investigating PLD2 levels throughout the menstrual cycle or at various stages of life, such as menopause, could also provide valuable insights. Additionally, investigating PLD2 levels in the context of various endocrine disorders, such as hypothyroidism, polycystic ovary syndrome, type 2 diabetes, and disorders associated with disruptions in sex hormone balance, such as primary ovarian insufficiency, could provide additional insights. Therefore, further research is needed to evaluate PLD2 expression in relation to sex and hormonal variations, with the goal of establishing specific reference values. This information could enhance its utility as a predictive and prognostic marker[19].
Sexual dimorphism
The differential expression of biomarkers based on patient sex is an increasingly recognized and critical area of medical research. A growing body of evidence has highlighted significant sex-based disparities in the incidence, pathogenesis, and clinical outcomes of pancreatitis[20-22]. These differences are likely influenced by sexual dimorphism[23,24] and sex-specific pathophysiological mechanisms, many of which involve cellular signaling pathways in which various biomarkers may play crucial roles. While some biomarkers have been studied in the context of pancreatitis, it is essential to acknowledge that the expression and functional activity of many biomarkers may vary significantly, depending on the patient’s sex. This underscores the importance of considering sex as a fundamental factor, not only in clinical diagnostics[20], imaging studies[25], and treatment, but also in the design of experimental studies. Failing to account for sex differences in research could limit our understanding of sex-specific disease mechanisms and hinder the development of targeted, sex-tailored therapeutic strategies.
Investigating the intricate interactions between sex hormones and the activity of biomarkers in pancreatic acinar and inflammatory cells could reveal both protective and exacerbating mechanisms that differ by sex, offering valuable insights into disease biology. Furthermore, this research may pave the way for the identification of novel sex-dependent biomarkers, thereby improving the precision of diagnostics and therapeutic interventions in pancreatitis. Incorporating sex as a critical variable in experimental design and biomarker discovery is essential for advancing personalized medicine in pancreatitis and other diseases with similar sex-based disparities. A more nuanced, sex-informed approach to research holds the potential to improve patient outcomes by facilitating the development of more effective, targeted diagnostic tools and therapeutic strategies[26].
Cost-effectiveness
The clinical implementation of any biomarker is primarily influenced by its cost-effectiveness. Wang et al’s study employed western blotting to measure PLD2, a method that presents significant limitations for routine clinical use, particularly in resource-limited settings[1]. These limitations include its technical complexity, long processing times, and high costs. For PLD2 to have a meaningful impact in clinical practice, a more accessible and cost-efficient approach is necessary. Western blotting, while reliable for research purposes, is ill-suited for large-scale diagnostic use, particularly in resource-limited settings. This technique is associated with high reagent costs, extended processing times and substantial labor and technical expertise requirements. These factors collectively hinder its scalability and feasibility for routine diagnostics. In contrast, enzyme-linked immunosorbent assay (ELISA) presents a more viable alternative for clinical application. ELISAs are characterized by lower reagent costs, faster turnaround times, and greater automation compatibility, making them more amenable to high-throughput testing. Furthermore, ELISA platforms are already widely used in clinical laboratories, which facilitates easier integration into existing diagnostic workflows.
Further cost reduction and diagnostic efficiency can be achieved through the development of multiplex assays. These assays allow simultaneous measurement of PLD2 along with other relevant biomarkers (e.g., inflammatory cytokines or cancer-related markers), thereby conserving sample volume, reducing processing time, and optimizing cost per analyte. Additionally, the use of automated platforms for assay processing can minimize human error, standardize results, and further lower long-term operational costs. Lastly, the emergence of point-of-care diagnostic technologies offers an innovative avenue for PLD2 detection. Lateral flow assays or portable biosensor-based formats, similar to those developed for procalcitonin or N-terminal pro-brain natriuretic peptide[27], could provide rapid, low-cost results directly at the bedside or in primary care settings. These platforms can be particularly transformative in low-resource or remote regions, where centralized laboratory infrastructure is lacking.
Advances in AP: Biomarkers and personalized diagnostic approaches
Despite the availability of numerous severity prediction systems for AP, such as Ranson, Acute Physiology and Chronic Health Examination II, systemic inflammatory response syndrome, and Bedside Index for Severity in AP, most exhibit comparable diagnostic performance and, importantly, generally low positive predictive values[28]. Traditional severity assessment tools, such as Ranson’s criteria and Acute Physiology and Chronic Health Examination II, though widely used, require substantial clinical and laboratory data, making them impractical for emergency settings where time-sensitive decisions are critical[29].
AP continues to pose a significant diagnostic challenge due to incomplete understanding of its pathophysiology and the absence of a universally validated biomarker[30]. AP pathophysiology is multifaceted, involving processes, such as damage-associated molecular patterns from necrotic acinar cells[31], genetic factors[32,33], epigenetic changes[34], immunogenic cell death[35], premature activation of trypsinogen, colocalization (fusion between lysosomes and zymogen granules within acinar cells)[36], disruptions in calcium signaling, autophagy, endoplasmic reticulum stress, ductal cell dysfunction, and dysregulation of microRNAs. It also involves various forms of cell death, such as apoptosis, necrosis, pyroptosis, and ferroptosis[37], pathogen-associated molecular patterns, the orointestinal microbiome[38,39], infections, the recruitment of immune cells, including neutrophils, macrophages, dendritic cells, and natural killer cells, as well as activation of inflammasomes[40]. All these processes play a key role in the inflammatory response. Moreover, the adaptive immune response, involving T cells, Treg cells, CD8+ cells, and B cells, further exacerbates the pathology of AP[41]. In addition to all this, new data on the role of the microbiota-gut-pancreas axis has emerged and has been postulated as a key player in the progression of pancreatitis severity. This, itself, has been considered a new potential therapeutic and diagnostic target[42,43]. A multi-omics approach may be instrumental in advancing our understanding of PLD2. Genomic analyses could uncover pathogenic variants, transcriptomic profiling may delineate expression patterns, proteomic studies can elucidate protein-protein interactions, and lipidomic data might clarify PLD2’s role in phospholipid signaling pathways. The integration of these complementary datasets would enable a more nuanced characterization of PLD2-related mechanisms, supporting its evaluation as a modifiable disease mediator, a prognostic or risk biomarker, or a potential indicator of treatment response.
Currently, serum amylase and lipase are the most commonly used biochemical markers, but their diagnostic reliability is hampered by institution-specific cutoff values. Lipase has demonstrated superior diagnostic sensitivity compared with amylase, particularly in biliary (97% vs 80%) and alcoholic pancreatitis (91%-100% vs 52%-55%), with high specificity (99.2% at 153 U/L). Lipase remains elevated for a longer duration, enhancing its clinical utility throughout the disease course, whereas amylase peaks early and declines rapidly, limiting its window of diagnostic relevance. Lipase also serves as a useful predictor of postoperative pancreatitis, whereas amylase demonstrates limited applicability in this context. Consequently, international guidelines now favor lipase over amylase, recommending context-adapted cutoff values[30]. However, a subset of patients with AP may present with normal enzyme levels, underscoring the need for caution, given that normal amylase and lipase levels do not rule out the diagnosis of AP[44].
In recent studies, PLD2 has emerged as a promising biomarker in the context of AP[1,3]. This enzyme plays a significant role in lipid metabolism and cell signaling, particularly in regulating inflammatory response. An altered expression of PLD2 in pancreatic tissues during acute and chronic stages of pancreatitis suggests its potential role in disease severity. Elevated levels of PLD2 have been associated with enhanced inflammation and could be a useful marker for predicting disease progression and guiding therapeutic decisions. As part of lipid metabolism, PLD2 is involved in modulating pathways that influence cell survival and immune activation, making it a candidate for inclusion in multimodal diagnostic approaches to AP.
In addition to enzyme-based markers, C-reactive protein (CRP) has been extensively studied and has shown good diagnostic accuracy. Nevertheless, CRP remains suboptimal for early prognostication, as neither admission CRP nor amylase levels reliably predict progression to severe disease[45]. To enhance early risk stratification, a creatinine-to-bilirubin ratio has demonstrated superior predictive capacity compared with individual markers. A creatinine-to-bilirubin ratio exceeding 14.05 is associated with higher rates of acute kidney injury, acute heart failure, increased 30-day mortality, and more frequent use of vasoactive and diuretic agents[46,47]. Additionally, biomarkers, such as interleukin-6 (IL-6), D-dimer, and serum calcium, measurable within six hours of admission, have shown promise in cases of hypertriglyceridemia-induced AP, offering ultra-early indicators for therapeutic guidance[48].
The neutrophil-to-lymphocyte ratio has emerged as a practical and accessible marker for distinguishing between infectious and sterile inflammation during the early stages of AP[29]. The triglyceride-glucose index has been proposed as a predictor of all-cause mortality in hospitalized patients with AP[49]. Similarly, leukotriene B4 has been proposed as a novel biomarker of severity, with significantly elevated levels in patients with necrotizing pancreatitis and correlations with amylase, lipase, and CRP[50]. Advances in molecular research have further highlighted potential biomarkers for this cause. For example, salusins (α and β), vasoactive peptides involved in vascular inflammation and oxidative stress, are elevated in severe AP, particularly salusin-β, which enhances inflammation via nuclear factor-κB signaling[51]. The von Willebrand factor-a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 axis is another promising marker, with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 showing potential in predicting pancreatic necrosis and persistent organ failure, whereas von Willebrand factor correlates with both[52]. Hypoalbuminemia (≤ 30 g/L) within the first 24 hours of admission affects one-third of AP patients[53] and is associated with systemic inflammatory response syndrome, pleural effusion, and increased mortality[54]. Albumin infusions are recommended in these patients, as they could significantly reduce mortality in cases of severe pancreatitis[55].
Recent advances in RNA biology have revealed the role of non-coding RNAs in AP. Long non-coding RNA small nucleolar RNA host gene 1, for instance, has shown an area under the curve of 0.89 in distinguishing AP from healthy controls, with experimental silencing reducing apoptosis and inflammation in pancreatic acinar cells[56]. Additionally, tRNA-derived small RNAs, such as tRF36, have been implicated in regulating pyroptosis and ferroptosis, potentially influencing disease progression[57].
Lipid metabolism is another emerging area of interest in AP. Patients with AP exhibit altered lipidomic profiles, including decreased levels of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid, alongside increased levels of phosphatidylinositol, and phosphatidylglycerol[58]. These changes suggest that lipids may modulate the inflammatory cascade in AP, in addition to serving as biomarkers. Other markers, such as fatty acid-binding protein 4, have been linked to poor survival outcomes[59], while IL-6 reached an area under the curve of 0.85 for diagnosing severe AP in a recent meta-analysis[60]. Mendelian randomization studies involve red blood cell count, leptin levels, blood glucose, and high leptin receptor sensitivity as potential causal factors in AP susceptibility[61]. Receptor-interacting protein kinase 3, a regulator of necroptosis, has been independently associated with disease severity and in-hospital delirium, suggesting its potential as both a biomarker and therapeutic target[62].
Technological innovations, such as hydrogel-based biosensors, offer promising new tools for non-invasive, highly sensitive detection of AP biomarkers. These biosensors, which boast high water content, adaptability, and biocompatibility, can detect a broad range of molecular targets, paving the way for earlier diagnosis and personalized management[63]. Furthermore, the use of artificial intelligence can enhance results by analyzing large volumes of data and identifying patterns beyond conventional biomarkers. However, as with other emerging biomarkers, further validation and standardization is required before their widespread clinical application[64].
While conventional enzyme-based diagnostics remain foundational in AP, a shift toward a multimodal diagnostic approach is emerging. This approach integrates clinical context, inflammatory and immune biomarkers, and molecular insights to more accurately assess disease severity and guide early therapeutic decisions. Novel biomarkers, such as urinary trypsinogen-2, phospholipase A2 and D2, and procalcitonin, alongside inflammatory cytokines like CRP, IL-6, and IL-8, can enhance diagnostic precision. Imaging techniques, such as contrast-enhanced computed tomography and magnetic resonance cholangiopancreatography, also play a crucial role in comprehensive evaluation[30]. Together, these integrated strategies potentially could improve outcomes in this complex and heterogeneous disease. Furthermore, acknowledging the influence of the ecto-exposome and endo-exposome[65] is critical for the advancement of personalized medicine.
Pancreatitis endotypes
Through transcriptomics, proteomics, and metabolomics, four distinct endotypes (A, B, C, D) with unique molecular characteristics have been identified. Endotype A is associated with greater severity and progression, characterized by markers, such as N-acetyl-3-methylhistidine, N-acetyl-1-methylhistidine, Xin actin-binding repeat-containing protein 1, and mitogen-activated protein kinase kinase kinase 6 (MAP3K6), and is linked to proteolysis, endothelial injury, and cell death. Endotype B, which includes homeobox D3, tripartite motif-containing 48, protein phosphatase 1 regulatory subunit 3A, and regenerating islet-derived protein 3A, is associated with cell adhesion, oxidative stress, cell death, and intestinal dysbiosis. Endotype C features gamma-glutamyl transferase 2, involved in glutathione homeostasis, and markers related to neurotransmission and sphingolipid biosynthesis. Finally, Endotype D is associated with pancreatic elastase 2 and Gilbert-type hyperbilirubinemia, linked to bile secretion. In addition, an inflammatory endotype has been described, characterized by elevated insulin, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin. Conversely, a non-inflammatory endotype has been identified, characterized by increased hepcidin levels[66]. This classification highlights the potential for tailored therapies and diagnosis based on molecular endotypes, paving the way for new biomarkers for pancreatitis[67].
Conclusion
PLD2 holds significant promise as a biomarker for assessing the inflammatory response in various pancreatic diseases, offering valuable insights for early diagnosis and monitoring. However, to fully realize its potential in clinical practice, it is essential to conduct multicenter studies with larger, more diverse sample sizes to further validate the findings and deepen our understanding of the underlying pathophysiological mechanisms. These studies should focus, not only on evaluating the diagnostic efficacy of PLD2, but also on assessing its cost-effectiveness, ensuring its accessibility in clinical settings. Ultimately, the integration of PLD2 into clinical practice could enhance personalized treatment approaches and improve outcomes for patients with pancreatic diseases. Further efforts are needed to narrow this research gap. It is also important to clarify whether PLD2’s role extends beyond simple correlation, to serve as a potential therapeutic target. Continued research in this area will be crucial for refining its role and maximizing its clinical impact.
