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

The research, conducted by an international team of investigators, presents a comprehensive comparative resource for adult human and mouse adrenal glands built on two complementary technologies: single-cell transcriptomics, which profiles the gene activity of thousands of individual cells, and spatial transcriptomics, which maps where those gene-expression programs reside within the intact architecture of the organ. By combining these approaches, the authors have produced what is effectively a high-resolution atlas of the adrenal gland in two species, capturing not only which cell types exist but also how their molecular identities and locations differ between human and mouse.

The significance of this resource lies in what it corrects. The adrenal cortex, the outer layer of the gland, is organized into zones that produce different classes of steroid hormones. In the classical textbook model, mineralocorticoids such as aldosterone are produced in the outermost zone, glucocorticoids such as cortisol in the middle zone, and androgen precursors in the innermost zone. This zonal model was largely established through work in rodents. The new single-cell and spatial data confirm that while broad zonal logic exists in both species, the underlying cellular states, the repertoire of expressed enzymes and the dynamics of cell differentiation diverge substantially between human and mouse, meaning that findings in mice cannot be assumed to apply directly to human adrenal physiology.

One of the study’s central findings concerns steroidogenic states. Steroid-producing cells do not represent a single, fixed identity; instead, they occupy a spectrum of molecular states defined by which steroidogenic enzymes they express, at what levels, and in what combinations. The comparative analysis shows that these states are shaped by species-specific programs. Human adrenal cells display enzymatic configurations and regulatory signatures that differ from their mouse counterparts in ways that affect how hormone synthesis is partitioned across the gland. For researchers studying disorders such as congenital adrenal hyperplasia, adrenal insufficiency, Cushing’s syndrome and primary aldosteronism, this is a critical caveat: therapeutic strategies validated in mouse models may engage cellular programs that human adrenal tissue either lacks or deploys differently.

The second major theme of the study is tissue turnover. The adrenal cortex is one of the most dynamic organs in the body, with its steroid-producing cells continuously replaced throughout life. The prevailing model, again derived largely from rodent studies, holds that a cap of progenitor cells near the outer surface of the gland continuously generates new steroidogenic cells that migrate inward, mature, perform their hormonal duties and eventually die, a conveyor-belt-like process of renewal. The new human data reveal that this turnover process, while conceptually conserved, proceeds with species-specific characteristics. The cellular sources of renewal, the differentiation trajectories and the pace of cell replacement differ between human and mouse, underscoring that the mouse conveyor-belt model is an approximation rather than a faithful replica of human adrenal maintenance.

Technically, the study’s strength comes from its dual-method design. Single-cell RNA sequencing excels at resolving cellular heterogeneity, distinguishing rare cell populations and reconstructing differentiation trajectories from the gene-expression fingerprints of individual cells. But dissociating an organ into single cells destroys spatial context, which is essential in an organ as architecturally organized as the adrenal gland. Spatial transcriptomics restores that context by measuring gene activity in intact tissue sections, allowing the researchers to verify that the cell states identified in the single-cell data occupy coherent anatomical positions. By applying both methods in parallel to human and mouse adrenal glands, the team could cross-validate cell-type assignments and map species differences with confidence, producing a resource designed to serve as a reference standard for the field.

The comparative framing also carries implications for regenerative medicine and drug development. Efforts to grow functional adrenal tissue in the laboratory, whether from stem cells or organoid cultures, depend on knowing which molecular programs must be activated to generate authentic steroid-producing cells. If those programs are species-specific, protocols optimized against mouse reference data may steer human cells toward the wrong developmental endpoints. Conversely, a human adrenal atlas provides a benchmark against which laboratory-grown adrenal cells can be quality-controlled, accelerating the path toward cell-based therapies for patients whose adrenal glands no longer function.

The resource is also expected to inform cancer research. Adrenocortical carcinoma is a rare but aggressive malignancy with limited treatment options, and its cellular origins remain incompletely understood. A detailed map of normal adrenal cell states in humans, including the progenitor populations and differentiation intermediates that tumors may hijack, offers researchers a framework for identifying which programs are reactivated in cancer and for designing therapies that target tumor-specific vulnerabilities while sparing normal steroid production.

Beyond disease, the study speaks to a broader lesson in modern biology: organ-level conclusions drawn from one species do not automatically transfer to another, even for organs as structurally similar as human and mouse adrenals. Evolution has conserved the gland’s essential function, the synthesis of life-sustaining steroids, but has implemented that function through partly divergent cellular and molecular means. As single-cell atlases accumulate across tissues and species, this theme recurs, and the adrenal gland now stands as a particularly clear example because its physiology is so directly tied to clinically essential hormones.

The authors have made their comparative dataset available as a community resource, allowing endocrinologists, developmental biologists and computational scientists to query cell-type markers, explore spatial gene-expression patterns and test their own hypotheses against the data. In an era when mouse models remain indispensable but increasingly scrutinized, resources of this kind provide the translational bridge that the field needs: a way to know precisely where the mouse is a faithful model of human biology, and where it is not. For the adrenal gland, at least, the answer is now written cell by cell, and it is more species-specific than anyone had fully appreciated.

Subject of Research: Comparative single-cell and spatial transcriptomic analysis of adult human and mouse adrenal glands

Article Title: Human and mouse adrenal glands are characterized by species-specific steroidogenic states and tissue turnover

Article References: Kastriti, M. E., Maksimov, D., Krupinova, J., Utkina, M., Beltsevich, D., Roslyakova, A., Glazova, O., Kaziakhmedova, S., Ryabova, A., Kuznetsova, A., Shcherbakova, A., Bondarenko, E., Antysheva, Z., Albert, E., Urusova, L., Lapshina, A., Chevais, A., Avsievich, E., Trofimov, V., … Adameyko, I. (2026). Human and mouse adrenal glands are characterized by species-specific steroidogenic states and tissue turnover. Nature Genetics, 58(9), 2270-2283. https://doi.org/10.1038/s41588-026-02737-1

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02737-1

Keywords: adrenal gland, single-cell transcriptomics, spatial transcriptomics, steroidogenesis, tissue turnover, human-mouse comparison, adrenal cortex, hormone production, cell atlas, Nature Genetics, endocrinology, regenerative medicine