Hormone-Driven Tumors Show Stronger Apelin Staining, First Study of Its Kind Finds

Deep inside the adrenal glands, small benign tumors can quietly flood the body with cortisol, producing the devastating constellation of symptoms known as Cushing’s syndrome. Now, a team of researchers in Turkey has taken one of the first steps toward understanding the molecular scenery inside these tumors, and their findings point to an unexpected player: apelin, a peptide best known for its roles in blood vessels and metabolism. In what the authors describe as the first study of its kind, tissue from cortisol-producing adrenal adenomas showed significantly stronger apelin staining than normal adrenal cortex, while the better-known blood vessel growth factor VEGF behaved in a surprisingly unremarkable way.

The research, conducted at Konya City Hospital and published in BMC Endocrine Disorders, examined archived tissue samples from eleven patients who underwent adrenalectomy for cortisol-producing adrenal adenomas between August 2020 and May 2025. These tumors are the most common cause of ACTH-independent Cushing’s syndrome, a form of the disease in which the adrenal glands secrete cortisol autonomously, ignoring the hormonal signals that normally keep production in check. Although endogenous Cushing’s syndrome remains rare, with an estimated annual incidence of 0.7 to 2.4 cases per million people, the consequences of prolonged cortisol excess, including cardiovascular disease, metabolic dysfunction, and elevated mortality, make understanding the biology of these tumors a pressing clinical concern.

To appreciate why the researchers focused on apelin and VEGF, it helps to understand the peculiar biology of the adrenal cortex. This outer layer of the adrenal gland is among the most richly vascularized endocrine tissues in the human body, a design feature that makes sense given its job: synthesizing steroid hormones and delivering them rapidly into the bloodstream. Angiogenesis, the formation of new blood vessels, is fundamental to both normal adrenal development and tumor growth, and VEGF has long been considered the master regulator of this process. Experimental evidence has linked increased VEGF signaling to higher microvascular density in adrenocortical tumors, suggesting that a tumor’s blood supply may be intertwined with its hormone output.

Apelin, by contrast, is a relative newcomer to this story. Discovered as the ligand of a G protein-coupled receptor called APJ, the apelin-APJ pathway is active throughout the body and participates in cardiovascular regulation, angiogenesis, and energy metabolism. Circulating apelin levels are known to rise in obesity, insulin resistance, and type 2 diabetes, conditions that overlap strikingly with the metabolic chaos of Cushing’s syndrome. Even more intriguing is the molecular cross-talk between the two systems: apelin expression can be induced under hypoxic conditions through hypoxia-inducible factor pathways, the same transcriptional network that controls VEGF, and experimental studies have shown that apelin can amplify VEGF-driven neovascularization through signaling cascades involving AMPK, PI3K/Akt, and eNOS.

The study’s methodology reflects the constraints of working with a rare disease. Because well-characterized surgical specimens from cortisol-producing adenomas are scarce, the researchers assembled a retrospective cohort of eleven tumor samples alongside fifteen control samples of histologically normal adrenal cortex, twelve derived from nephrectomy specimens and three from tissue adjacent to benign adrenal lesions. To ensure that only benign tumors were included, every specimen was evaluated with the Weiss scoring system, a nine-parameter histopathological algorithm that distinguishes adenomas from carcinomas; only tumors with a Weiss score of two or below made the cut. Formalin-fixed, paraffin-embedded sections were stained for apelin and VEGF using an automated immunohistochemistry platform, with placental tissue serving as the positive control and omission of the primary antibody providing the negative control.

Scoring was deliberately rigorous. Three experienced pathologists, blinded to all clinical data and histopathological parameters, independently assessed each slide, grading both the percentage of positively stained cells and the staining intensity on semi-quantitative scales. The product of these two values yielded an immunoreactivity score ranging from zero to twelve. Interobserver agreement was impressively high, with pairwise weighted Cohen’s kappa coefficients for apelin staining intensity ranging from 0.889 to 0.927, indicating near-perfect concordance among the evaluators. Recognizing the statistical fragility inherent in such a small cohort, the team also deployed resampling-based methods, including permutation testing with 10,000 iterations, bootstrap confidence intervals built from 5,000 resamples, and bias-reduced logistic regression to guard against small-sample instability.

The headline result was a difference in staining intensity rather than quantity. The proportion of apelin-positive cells was essentially identical between tumors and controls, at roughly 29 percent versus 31 percent. But the intensity of that staining told a different story: strong apelin staining appeared in five of the eleven tumor samples, or 45.5 percent, while not a single control tissue showed strong staining. This difference reached statistical significance with a p value of 0.026 and a large effect size, and it held up in a sensitivity analysis restricted to nephrectomy-derived controls. An exploratory receiver operating characteristic analysis found that apelin staining intensity discriminated between tumor and normal tissue with moderate accuracy, yielding an area under the curve of 0.776, and bias-reduced logistic regression associated higher staining intensity with tumor status with an odds ratio of 2.96, though the wide confidence interval, spanning 1.02 to 8.60, signals considerable uncertainty.

VEGF, the anticipated star of the angiogenic show, refused to cooperate. None of its measured parameters, including the percentage of stained cells, staining intensity, or the composite immunoreactivity score, differed significantly between tumors and controls. The authors interpret this cautiously, suggesting that VEGF expression in the adrenal cortex may simply reflect the tissue’s intrinsically rich vascular architecture rather than any tumor-specific alteration. Consistent with this, no significant correlation emerged between apelin and VEGF immunoreactivity scores within the tumor group, meaning the study found no direct immunohistochemical evidence of the apelin-VEGF cross-talk documented in experimental systems. Nor did either marker correlate with tumor size, Ki-67 proliferation index, mitotic count, or any of the hormonal measurements, including ACTH, morning cortisol, midnight cortisol, and 24-hour urinary free cortisol.

The authors are admirably candid about the limits of their findings. With eleven patients, the study was exploratory by necessity, and multiple statistical comparisons were performed without formal adjustment for multiplicity, raising the possibility that the significant apelin result is a nominal finding that could evaporate upon replication. The retrospective design precluded verification of urine collection completeness and systematic assessment of anthropometric and metabolic parameters, leaving open the tantalizing but untested question of whether stronger apelin staining relates to the metabolic fallout of cortisol excess. Residual confounding by sex also cannot be excluded, since women predominated in the tumor group. And because apelin was measured in postoperative histological specimens, the discriminatory power observed in the ROC analysis says nothing about clinical diagnostic utility; no one is proposing apelin staining as a diagnostic test.

Still, the study opens a genuinely new window. Prior work on angiogenesis in adrenal tumors has been sparse and sometimes contradictory: one study found higher expression of the endothelial marker endocan in cortisol-producing adenomas, another reported elevated CD31 in adrenocortical carcinoma, and VEGF findings across the literature have been heterogeneous. The only previous examination of apelin in adrenal-related tumors found reduced expression in metastatic pheochromocytomas and paragangliomas, tumors of the adrenal medulla rather than the cortex, underscoring how different tumor lineages may behave. What happens next will require larger multicenter cohorts, direct measures of angiogenesis such as microvessel density and endothelial markers, and molecular tools capable of quantifying apelin, its APJ receptor, and VEGF receptors beyond the semi-quantitative lens of immunohistochemistry. For now, the message is modest but real: inside these cortisol-secreting tumors, something about apelin looks different, and figuring out why may illuminate how hormone production, metabolism, and blood vessel growth intertwine in one of medicine’s most quietly destructive endocrine diseases.

Subject of Research: Apelin and VEGF immunohistochemical expression in cortisol-producing adrenal adenomas associated with Cushing's syndrome

Article Title: Immunohistochemical evaluation of apelin and VEGF expression in cortisol-producing adrenal adenomas

Article References: Çalişkan Burgucu, H., Günler, T., Ömeroğlu, E., Ünlü, Y., & Aksu, O. (2026). Immunohistochemical evaluation of apelin and VEGF expression in cortisol-producing adrenal adenomas. BMC Endocrine Disorders, 26(1), Article 274. https://doi.org/10.1186/s12902-026-02563-w

Image Credits: AI Generated

DOI: 10.1186/s12902-026-02563-w

Keywords: cortisol-producing adrenal adenoma, Cushing's syndrome, apelin, VEGF, angiogenesis, immunohistochemistry, adrenal cortex, endocrine tumors, hypercoritsolism, APJ receptor, metabolism, histopathology