Letter to the Editor: Exosomal circular RNAs from peripheral blood-derived mesenchymal stem cells as diagnostic and subtyping biomarkers in acute mesenteric ischemia

Abstract

The study evaluates the diagnostic utility of peripheral blood-derived mesenchymal stem cell exosomal circular RNAs (circRNAs) in acute mesenteric ischemia (AMI). In a cohort of 80 AMI patients and 125 controls, Wang et al found that circ-Eya3 was significantly upregulated, while circEZH2_005 was downregulated in AMI patients. These biomarkers strongly correlated with intestinal injury markers (intestinal fatty acid binding protein, D-lactate) and systemic inflammation (neutrophil-to-lymphocyte ratio, interleukin-6). The integration of these circRNAs with D-lactate yielded high diagnostic accuracy (area under the curve = 0.891), suggesting their preliminary utility as adjunct markers for the early detection and characterization of AMI.

Key Words: Acute mesenteric ischemia; Peripheral blood-derived mesenchymal stem cells; Exosomal circular RNAs; circ-Eya3; circEZH2_005

Core Tip: Peripheral blood-derived mesenchymal stem cell exosomal circ-Eya3 and circEZH2_005 represent a promising step toward a ‘liquid biopsy’ approach for acute mesenteric ischemia. Beyond simple diagnosis, their differential expression patterns showed a statistically significant variance between arterial and venous etiologies in this cohort, suggesting a potential role in etiologic differentiation that may help signal the critical transition from early reversible ischemia to advanced necrosis.

TO THE EDITOR

The clinical management of acute mesenteric ischemia (AMI) is a race against time, where the window between reversible ischemia and irreversible transmural necrosis is narrow. Current diagnostic efforts are hampered by the lack of specific biomarkers, as traditional indicators of acute abdominal pain are non-specific. The identification of peripheral blood-derived mesenchymal stem cell (PBMSC) exosomal circular RNAs (circRNAs) addresses these diagnostic hurdles[1] through the following expanded pillars.

Superior biological stability for liquid biopsy

circRNAs represent a significant advanceover linear RNA biomarkers due to their unique covalent closed-loop structure.

RNase resistance: Unlike linear RNAs, which have 5’ and 3’ ends susceptible to exonucleolytic degradation, circRNAs are highly resistant to RNase[2].

Extended half-life: As highlighted by the investigators, this structural integrity provides circRNAs with an extended half-life, making them highly abundant and reliable targets for liquid biopsies in clinical settings[3].

Diagnostic integrity: Their inherent stability ensures that the molecular signature remains intact from the time of blood draw to analysis, providing a consistent reflection of the patient’s pathological state[4].

Dual reflectivity: Intestinal injury and systemic inflammation

The utility of these markers lies in their ability to offer a “holistic” view of the patient’s condition, bridging the gap between localized tissue damage and the body’s systemic reaction.

Indicator of enterocyte damage: circ-Eya3 and circEZH2_005 are strongly correlated with established injury markers. Specifically, circ-Eya3 positively correlates with intestinal fatty acid binding protein (IFABP) and D-lactate, which are classic indicators of enterocyte damage and barrier malfunction[5].

Indicator of inflammatory burden: Beyond physical damage, these circRNAs reflect the systemic inflammatory response. circ-Eya3 positively correlates with interleukin-6 (IL-6) and the neutrophil-to-lymphocyte ratio (NLR), while circEZH2_005 shows a strong inverse relationship with these indices[6].

Comprehensive risk stratification: This dual reflectivity allows clinicians to assess not just that the intestine is failing, but also the severity of the associated systemic inflammatory storm[7].

Precision subtype and stage guidance

One of the most critical challenges in AMI is determining the etiology and degree of necrosis, as these dictate the immediate intervention strategy.

Differentiating arterial vs venous AMI: Traditional biomarkers like intestinal fatty acid binding protein (IFABP) can be inconsistent. However, circEZH2_005 expression was distinct between arterial occlusion and venous thromboembolism within the study group, warranting further validation (requiring revascularization) and venous thromboembolism (prioritizing anticoagulation)[8].

Identifying surgical urgency: The expression patterns of these circRNAs diverge between early reversible ischemia and advanced necrotic AMI. For instance, circ-Eya3 is significantly upregulated in late necrotic stages compared with early stages[9].

Guiding targeted intervention: These specific expression patterns may provide supportive data for subtyping and staging, which could eventually assist in triaging patients and minimizing delays in vascular consultation and avoiding unnecessary or misdirected surgeries[9]. These properties position circRNAs as robust diagnostic biomarkers with potential for clinical triage (Figure 1).

Figure 1 Diagnostic utility of peripheral blood-derived mesenchymal stem cell-exosomal circular RNAs in acute mesenteric ischemia. The clinical significance of peripheral blood-derived mesenchymal stem cell exosomal circular RNAs is rooted in their covalent closed-loop structure, which provides inherent stability and resistance to RNase-mediated degradation compared with linear mRNA. These biomarkers function as dual indicators, simultaneously reflecting the severity of enterocyte damage and the systemic inflammatory burden associated with acute mesenteric ischemia. Furthermore, their differential expression patterns allow for precise surgical triage by distinguishing early reversible ischemia from advanced necrotic acute mesenteric ischemia, thereby identifying critical surgical urgency. Created in BioRender (Supplementary material). MSCs: Mesenchymal stem cells; circRNAs: Circular RNAs; AMI: Acute mesenteric ischemia.

METHODOLOGICAL STRENGTHS

We read the study by Wang et al[1] with great interest, which utilized a robust triple-validation framework for exosome characterization, confirming the characteristic cup-shaped morphology via transmission electron microscopy, a size range of 35-100 nm through nanoparticle tracking analysis, and the presence of tumor susceptibility gene 101 and CD63 surface markers via western blot. This rigorous approach, combined with the classification of 80 AMI patients into early reversible and late necrotic subgroups, provides a nuanced understanding of biomarker behavior. Furthermore, the inclusion of 125 symptomatic non-AMI controls ensures the specificity of the circRNA-based diagnosis against other acute abdominal conditions. A summary of exosomal characterization parameters and diagnostic performance is provided in Table 1.

Table 1 Summary of peripheral blood-derived mesenchymal stem cell-exosomal biomarker characterization and performance.

Parameter	Method/marker	Key findings/values
Morphology	TEM	Characteristic cup-shaped vesicles
Size distribution	NTA	35-100 nm diameter range
Surface markers	Western blot	Positive for TSG101 and CD63
Diagnostic accuracy	circ-Eya3	AUC = 0.677
Diagnostic accuracy	circEZH2_005	AUC = 0.741
Combined panel	Triple-marker	AUC = 0.891

TEM: Transmission electron microscopy; NTA: Nanoparticle tracking analysis; TSG101: Tumor susceptibility gene 101; AUC: Area under the curve.

NOVEL INSIGHTS OFFERED BY THE STUDY

The study provides several pioneering observations that advance the molecular understanding of AMI diagnosis.

Superiority of the multi-marker approach

While individual biomarkers show promise, the study demonstrates that the diagnostic power of these biomarkers is optimized through their strategic combination.

Limitations of single markers: Individual analysis of circ-Eya3 [area under the curve (AUC) = 0.677], circEZH2_005 (AUC = 0.741), and D-lactate (AUC = 0.787) shows only moderate predictive value.

Synergistic accuracy: The integration of these two novel circRNAs with traditional D-lactate significantly elevates diagnostic precision, achieving an excellent AUC of 0.891.

Addressing heterogeneity: This multi-marker strategy effectively addresses the limitations of single-biomarker reliance, such as the heterogeneity of IFABP levels in advanced necrosis, which can lead to diagnostic ambiguity.

Specific biomarker polarization as internal validation

A unique and clinically valuable insight offered by the study is the divergent expression patterns of the two primary circRNAs.

Polarized expression: In AMI patients, circ-Eya3 was significantly upregulated while circEZH2_005 was downregulated compared with healthy controls.

Built-in verification: This “mirror-image” behavior, where one marker increases and the other decreases, provides a built-in internal validation for clinical assessment.

Disease correlation: The study reveals that this polarization extends to secondary indices; circ-Eya3 positively correlates with inflammatory markers (NLR, IL-6), whereas circEZH2_005 shows a strong inverse relationship with these same indices.

Reperfusion sensitivity and monitoring

The study introduces a potential new metric for evaluating the success and impact of surgical interventions.

Post-operative “peak”: Following revascularization or embolectomy, circ-Eya3, IFABP, and D-lactate levels exhibit a sharp, significant increase.

Ischemia-reperfusion injury metric: This immediate post-operative peak in circ-Eya3 warrants further investigation as a supplementary metric for assessing the severity of ischemia-reperfusion injury.

Dynamic utility: The markers’ immediate post-intervention fluctuations offer clinicians a window into real-time intestinal recovery or damage, facilitating more precise post-surgical monitoring.

While the immediate post-operative increase in circ-Eya3 levels correlates with the timing of revascularization, it is important to acknowledge that this ‘peak’ is likely multifactorial. Beyond ischemia-reperfusion injury, the physiological stress of anesthesia, surgical trauma from intestinal resection, and the systemic inflammatory response to major abdominal surgery may independently stimulate the release of PBMSC-exosomal circRNAs. Therefore, clinicians should exercise caution to avoid false-positive interpretations of these elevations, treating them as markers of general perioperative stress rather than isolated indicators of reperfusion severity until more specific comparative studies are conducted.

The diagnostic performance of the triple-marker panel (AUC = 0.891) should be viewed as a complementary tool to existing clinical standards. Unlike generalized scoring systems such as APACHE II or SOFA, which reflect systemic multi-organ failure, the PBMSC-exosomal circRNA panel offers high specificity for intestinal barrier dysfunction. Furthermore, while computed tomography angiography is the definitive method for identifying macrovascular occlusion, these circRNAs may offer a ‘biological’ advantage by identifying early reversible ischemia or microvascular reperfusion injury before gross anatomical changes are visible on standard imaging. Future studies should prioritize head-to-head comparisons of these biomarkers against the sensitivity and negative predictive values of gold-standard imaging to better define their role in the emergency triage algorithm.

POINTS FOR CLARIFICATION/OPPORTUNITIES FOR FUTURE WORK

While the results of this study are promising, there are several areas for further exploration to ensure these findings can be successfully translated into routine clinical practice.

Elucidation of mechanistic pathways

Although a strong correlation has been established between these circRNAs and the inflammatory burden of AMI, the underlying biological pathways remain largely hypothetical.

Intracellular signaling: Further investigation is required to determine how PBMSC exosomal circ-Eya3 and circEZH2_005 interact with target cells, such as macrophages or intestinal epithelial cells, to modulate systemic inflammation.

Functional modeling: Future research should utilize in vitro cell assays and in vivo animal models to confirm whether these circRNAs are active drivers of tissue repair or merely passive markers of injury.

Metabolic implications: Given that circ-Eya3 is associated with ATP production under glucose deficiency, its specific role in helping intestinal cells survive acute hypoxia deserves deeper molecular profiling[10].

Standardization of reference ranges and isolation

To move from a research setting to a diagnostic standard, the variability inherent in exosome research must be addressed.

Multicenter validation: Large-scale, multicenter cohorts are essential to define standardized reference ranges for circ-Eya3 and circEZH2_005 across diverse patient populations.

Pre-analytical consistency: Standardizing protocols for blood collection, plasma processing, and storage is critical to minimize variability in circRNA yield.

Comparative isolation: While ultracentrifugation is the gold standard for research, future studies should compare these results with more clinically feasible isolation methods, such as polymer-based precipitation or immunoaffinity capture[11].

Accelerated clinical translation

The “gold standard” for AMI diagnosis must be speed, as every hour of delayed revascularization increases the risk of mortality and short bowel syndrome.

Rapid detection platforms: Future work should focus on integrating circRNA detection with rapid diagnostic tools, such as digital polymerase chain reaction or point-of-care testing devices, to provide results within the critical “golden hour”.

Microfluidic integration: Developing “exosome-on-a-chip” devices could streamline the workflow from isolation to quantification, meeting the urgent need for instant diagnosis in emergency departments.

Predictive algorithms: Integrating these circRNA levels into existing clinical scoring systems could enhance the ability of vascular surgeons to triage patients for immediate revascularization vs conservative anticoagulation[12].

Pathways to clinical translation

The transition of PBMSC-exosomal circRNAs into routine emergency medicine faces two primary challenges: Isolation speed and mechanistic clarity. Currently, the isolation process relies on ultracentrifugation, which is geographically and temporally restricted. To achieve true clinical utility, further efforts must integrate these biomarkers with automated microfluidic ‘exosome-on-a-chip’ devices capable of delivering results within the critical diagnostic window. Furthermore, while the current data establishes a robust ‘liquid biopsy’ signature, the underlying biological mechanisms remain to be fully elucidated. Specifically, investigating how circ-Eya3 and circEZH2_005 modulate the systemic inflammatory storm, potentially through target cell interaction with macrophages or intestinal epithelial cells, is essential to determine whether these markers are active therapeutic targets or passive indicators of injury. The potential clinical applications discussed herein should be interpreted with caution, as they are based on preliminary findings from a single-center study and require further validation in larger, independent cohorts before clinical implementation. The current evidence is limited by the absence of comprehensive comparisons with established diagnostic standards, insufficient evaluation of diagnostic performance, and potential biases inherent in the study design, thereby restricting immediate clinical applicability.

Limitations of the study

While the findings regarding PBMSCs exosomal circ-Eya3 and circEZH2_005 are promising, several limitations must be acknowledged to contextualize their clinical readiness.

Sample size and study design: The study relied on a relatively small cohort of 80 AMI patients. The single-center nature of the research may limit the generalizability of these biomarkers across more diverse, global patient populations.

Lack of external validation: Although the diagnostic accuracy (AUC = 0.891) is high within this specific group, independent external validation in separate cohorts is required to confirm these results.

Technical challenges in standardization: The use of sequential ultracentrifugation, while a research gold standard, is time-consuming and difficult to scale for emergency department use. Standardizing pre-analytical variables, such as blood processing and storage, remains a significant hurdle for achieving consistent circRNA yields.

Mechanistic uncertainty: The study primarily establishes a correlation between these circRNAs and AMI pathology. However, the exact intracellular signaling pathways and functional roles of these molecules in tissue repair vs injury remain largely hypothetical.

Detection speed: To meet the “golden hour” requirement for AMI treatment, current detection methods like reverse transcription real-time polymerase chain reaction must be transitioned to more rapid, point-of-care platforms to be clinically actionable.

CONCLUSION

The clinical management of AMI is often a race against time, hampered by a lack of specific biomarkers and non-specific clinical symptoms that lead to high mortality and severe morbidity. These markers provide “dual reflectivity”, mirroring both the physical severity of enterocyte damage through correlations with IFABP and D-lactate, and the systemic inflammatory burden via correlations with IL-6 and NLR. A pioneering insight offered by this research is the multi-marker approach; while individual circRNAs show moderate predictive value, their combination with D-lactate achieves excellent diagnostic accuracy (AUC = 0.891). This specific biomarker polarization, where circ-Eya3 is upregulated and circEZH2_005 is downregulated, provides a built-in internal validation for clinical assessment. Furthermore, the immediate post-operative “peak” in circ-Eya3 offers a potential new metric for assessing the severity of ischemia-reperfusion injury in real-time. Ultimately, integrating these PBMSC-exosomal circRNAs into clinical practice could significantly reduce diagnostic inertia and improve patient survival by shortening the critical time to vascular consultation.