Introduction

Glucocorticoid (GC) replacement therapy, typically administered orally through agents such as GC-like hydrocortisone or prednisolone, plays a pivotal role in managing adrenal insufficiency. However, achieving optimal GC replacement poses challenges, with long-term administration potentially suppressing the hypothalamus–pituitary–adrenal axis, leading to immunosuppression, diabetes mellitus, dyslipidemia, and osteoporosis1. Cell therapy, involving the transplantation of regenerative steroid-producing cells, has emerged as a potential strategy to restore hormone responses to physiological stimuli2,3, and studies focused on generating steroid-producing cells from stem cells have been reported4,5,6,7,8,9,10,11,12,13.

We previously generated steroid-producing cells through the targeted overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), also known as Steroidogenic factor 1/Adrenal 4-binding protein (SF-1/Ad4BP), which is a master regulator of steroid production14,15,16,17. The implantation of NR5A1-induced steroidogenic cells extended the survival of adrenal insufficient mice18,19. However, the implantation of NR5A1-induced steroidogenic cells derived from mesenchymal stromal cells into adrenocortical cells is inadequate because of their characteristic secretion of both adrenal and gonadal steroids5,7,8,18,19,20. Further induction of differentiation is necessary to obtain steroid cells specialized for glucocorticoid replacement.

The adrenal cortex exhibits a dense vascular network characterized by fenestrated endothelial cells21,22,23,24. This vasculature is integral for oxygen and nutrient delivery as well as steroid hormone transport. Studies have demonstrated its crucial involvement in adrenal growth and differentiation during embryogenesis, providing the requisite precursors for steroid hormone biosynthesis and facilitating bloodstream secretion22,25,26,27,28. Functional interactions between adrenocortical and vascular endothelial cells (VECs) suggest that VECs may regulate steroid production. Specifically, adrenal capillary endothelial cells enhance aldosterone production29,30. In this study, to determine whether VECs can differentiate NR5A1-induced steroidogenic cells into more adrenocortical-like cells, we co-cultured NR5A1-induced steroidogenic cells with VECs and examined their effects on steroidogenesis.

Results

Expression levels of NR5A1 and VEGFA in adeno-NR5A1-infected ADSCs

To generate NR5A1-induced steroidogenic cells, ADSCs were isolated from mouse subcutaneous adipose tissue and infected with NR5A1-expressing adenovirus (moi = 25). Adeno-lacZ-infected ADSCs served as controls. On day 3 post-infection, the DsRed positive percentage in NR5A1-ADSCs was 45.9 ± 4.3%. Furthermore, immunostaining revealed NR5A1 expression localized to the nuclei of DsRed-positive cells, and western blotting confirmed a significant increase in NR5A1 protein levels (Fig. 1A,B). As VEGFA secreted by adrenocortical cells promotes angiogenesis, we investigated whether VEGFA was induced in NR5A1-ADSCs. Compared to ctrl-ADSCs, Vegfa mRNA expression was significantly increased in NR5A1-ADSCs. Hif1a, a transcriptional regulator of Vegfa and activator of hypoxic signaling, exhibited increased expression in NR5A1-ADSCs (Fig. 1C). The secretion of VEGFA consistently and significantly increased (Fig. 1D).

Figure 1
figure 1

NR5A1 and VEGFA expressions in adeno-NR5A1-inoculated ADSCs. (A) Structure of NR5A1-expressing adenos, lacZ was used as control, and DsRed as the infection confirmation label. Three days after adenovirus infection, ADSCs were subjected to immunostaining with anti-NR5A1 antibody. Scale bar = 50 µm. (B) Expression of NR5A1 in ADSCs on day 3 after adenovirus infection was analyzed using western blotting. A 5 µg of protein was loaded on each lane. NR5A1 and HSP90 (internal control) intensity was measured, and NR5A1 expression was graphed. (C) Vegfa and Hif1a expression levels were analyzed using qPCR. (D) VEGFA concentration of culture medium was measured using ELISA. Student’s t-test, *p < 0.05, ***p < 0.001. ADSCs, adipose-derived stem cells; DsRed, Discosoma sp. red fluorescent protein ELISA; enzyme-linked immunosorbent assay; HSP90: heat shock protein 90; NR5A1, nuclear receptor subfamily 5 group A member, VEGFA, vascular endothelial growth factor A.

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Co-culture with VECs promoted corticosterone but not testosterone production in NR5A1-induced steroidogenic cells

Immunostaining of mouse adrenal glands revealed NR5A1-positive cells adjacent to vascular endothelial cells, as previously reported (Supplementary Fig. 1)24,25. Subsequently, we analyzed the effect of VECs on the steroid production of NR5A1-ADSCs. ADSCs were infected with adenovirus and cultured for 3 days, followed by co-culture with mouse VECs for 4 days (Fig. 2A). Corticosterone and testosterone levels in the NR5A1-ADSC medium were measured and compared in the absence and presence of co-culture with VECs. The findings revealed that corticosterone production significantly increased depending on the number of VECs. However, co-culture with VECs did not alter testosterone production, resulting in an increased corticosterone/testosterone ratio (Fig. 2B). Cortisol produced by NR5A1-ADSCs increased significantly compared to ctrl-ADSCs but was not altered by co-culture with VECs (Fig. 2C). Aldosterone production in NR5A1-ADSCs was not significantly different from that in ctrl-ADSCs and was not altered by co-incubation with VECs (Fig. 2C).

Figure 2
figure 2

Co-culture with VECs enhanced corticosterone secretion from NR5A1-induced steroidogenic cells. (A) Co-culture procedure overview. ADSCs (5.0 × 104 cells/well) were inoculated with adeno-lacZ (Ctrl) or adeno-NR5A1 (NR5A1) (MOI = 25). Three days after inoculation, 5.0 × 104 NR5A1-ADSCs were co-cultured with 0, 5 × 103, 2.5 × 104, or 5 × 104 of VECs, denoted as NR5A1, NR5A1:VEC = 1:0.1, NR5A1:VEC = 1:0.5, or NR5A1:VEC = 1:1, respectively. Subsequently, they were co-cultured for 4 days. (B) Corticosterone, and testosterone levels in the medium were measured using ELISA, and the ratio of corticosterone to testosterone (B/T) was calculated. (C), Ctrl-ADSCs, NR5A1-ADSCs, and NR5A1-ADSCs + VEC were cultured similarly, and cortisol and aldosterone were measured using LC–MS/MS. (D), Phase contrast images showing the morphology of NR5A1-ADSCs (upper panel) and NR5A1-ADSCs (lower panel) co-cultured with VECs. DsRed positive cells in each condition were counted and normalized by region of interest (n = 3, mean ± standard error). Statistical analyses were performed using Student’s t-test or one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ns; not significant). Scale bar = 100 μm. ADSCs, Adipose-derived stem cells; ANOVA, Analysis of variance; DHEA, Dehydroepiandrosterone; DsRed, Discosoma sp. red fluorescent protein; ELISA, enzyme-linked immunosorbent assay; LC–MS/MS, liquid chromatography-tandem mass spectrometry; MOI, multiplicity of infection; NR5A1, Nuclear receptor subfamily 5 group A member 1; VEC, vascular endothelial cells.

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The morphology of NR5A1-ADSCs (DsRed-positive cells) co-cultured with VECs appeared reduced and smaller than that of NR5A1-ADSCs cultured alone (Fig. 2D). Nonetheless, no significant difference was observed in the number of DsRed-positive cells, indicating that co-culture elicited no effect on cell growth in NR5A1-ADSCs.

Expression levels of steroidogenic enzymes in NR5A1-ADSCs co-cultured with VECs

Considering the observed increase in corticosterone production in NR5A1-ADSCs co-cultured with VECs, we investigated the expression levels of steroidogenic enzymes. To isolate only NR5A1-ADSCs from the co-culture with VECs, DsRed-positive cells were sorted using a cell sorter. Controls comprised NR5A1-ADSCs without co-culture (Fig. 3A). The qPCR results revealed significant increases in the expression levels of steroidogenic acute regulatory protein (Star), cytochrome P450 family 11 subfamily a member 1 (Cyp11a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1), cytochrome P450 family 21 subfamily a member 1 (Cyp21a1), and cytochrome P450 family 11 subfamily b member 1 (Cyp11b1) in NR5A1-ADSCs co-cultured with VECs, when compared with NR5A1-ADSCs without co-cultures (Fig. 3B). The expression levels of cytochrome P450 family 17 subfamily a member 1 (Cyp17a1), hydroxysteroid 17-b dehydrogenase 3 (Hsd17b3), and cytochrome P450 family 19 subfamily a member 1 (Cyp19a1) remained unchanged (Fig. 3B).

Figure 3
figure 3

Expression levels of steroidogenic enzymes in NR5A1-ADSCs co-cultured with VECs. (A) ADSCs cultured for 3 days after adeno-infection were co-cultured with VECs for 4 days, and DsRed positive cells were sorted. (B) Expression levels of steroidogenic enzymes in the sorted cells were analyzed using qPCR. (C) Expression levels of Fdxr, Fdx1, Por, and Cyb5a were analyzed using qPCR. (D) Enzymes and electron transfer systems in the pathway of biosynthesis of corticosterone and aldosterone from cholesterol. Statistical analysis was performed using the Student’s t-test (*p < 0.05, **p < 0.01, ns; not significant). ADSCs, adipose-derived stem cells; Cyb5a, cytochrome b5 type A; DsRed, Discosoma sp. red fluorescent protein; Fdx1, ferredoxin 1; Fdxr, ferredoxin reductase; Por cytochrome P450 oxidoreductase; VEC, vascular endothelial cells; qPCR, quantitative polymerase chain reaction.

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Mitochondrial P450s such as Cyp11a1, Cyp11b1, and Cyp11b2 received electrons from Ferredoxin reductase (Fdxr) and Ferredoxin 1 (Fdx1), whereas Cyp17a1, Cyp21a1, and Cyp19a1, localized in the endoplasmic reticulum, are P450 oxidoreductase (Por) dependent. The 17- and 20-lyase activities of Cyp17a1 rely on the hemoprotein cytochrome b5 type a (Cyb5a). Considering the potential contribution of the electron transfer system in increasing corticosterone production, we examined the expression levels of Fdrx, Fdx1, Por, and Cyb5a. Co-culture with VECs significantly increased the expression of Fdxr, Por, and Cyb5a but not Fdx1. Despite the induction of Cyb5a expression by co-culture, the lack of increased testosterone production did not appear to be attributed to reduced Cyp17a1 activity owing to decreased Cyb5a levels (Fig. 3C). The enzymes and electron transfer systems involved in corticosterone production are shown in Fig. 3D. The expression of these genes in NR5A1-ADSCs was increased by co-culture with VECs.

Co-culture with VEC increased the mRNA expression of Mc2r and Mrap in NR5A1-ADSC

The production of glucocorticoids in mammals is regulated by adrenocorticotropic hormone (ACTH), which is secreted by the pituitary gland. The ACTH receptor is encoded by the Melanocortin 2 receptor (Mc2r). The Mc2r accessory protein Mrap is a protein that is necessary for the transport of the ACTH receptor to the cell membrane and for ACTH receptor signaling31. NR5A1-ADSCs co-cultured with VEC were sorted, and real-time PCR was performed. The expression of Mc2r and Mrap in NR5A1-ADSCs was significantly increased by co-culture with VEC (Fig. 4A). The gonadotropin receptors luteinizing hormone/choriogonadotropin receptor (Lhcgr) and follicle-stimulating hormone receptor (Fshr) were not detected in NR5A1-ADSCs or NR5A1-ADSCs co-cultured with VEC.

Figure 4
figure 4

Expression levels of Mc2r and Mrap in NR5A1-ADSCs co-cultured with VECs. (A) ADSCs cultured for 3 days after adeno-infection were co-cultured with VECs for 4 days, and DsRed positive cells were sorted. Ctrl-ADSCs were adeno-lacZ inoculated ADSCs. Expression levels of Mc2r, Mrap, Lhgcr, and Fshr were analyzed using qPCR. Lhgcr and Fshr were not detected using qPCR. Statistical analysis was performed using one-way ANOVA (**p < 0.01, ns; not significant). (B) 2 × 104 ADSCs were infected with an adenovirus vector. On the third day after the infection, 2 × 104 VECs were added for co-culture, and on the fifth day after the infection, the medium was replaced with medium containing 2 µM ACTH or 10 µM forskolin and incubated for another 2 days. The concentration of corticosterone in the medium was measured. Statistical analysis was performed using a one-way ANOVA. ns; not significant. ACTH, adrenocorticotropic hormone; ADSCs, adipose-derived stem cells; Agtr1, angiotensin II receptor type 1; DsRed, Discosoma sp. red fluorescent protein; Fshr, follicle-stimulating hormone receptor; Lhcgr, luteinizing hormone/choriogonadotropin receptor; Mc2r, melanocortin 2 receptor; Mrap, melanocortin 2 receptor accessary protein; NR5A1, nuclear receptor subfamily 5 group A member 1; VEC, vascular endothelial cells; qPCR, quantitative polymerase chain reaction.

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As the expression of Mc2r and Mrap increased when co-cultured with VEC, we stimulated the cells with ACTH or forskolin to investigate their ACTH responsiveness. NR5A1-ADSCs were stimulated with ACTH or forskolin for 48 h, and the concentration of corticosterone in the culture medium was measured. No increase in corticosterone was observed with ACTH stimulation. Forskolin tended to increase the corticosterone levels, but no significant increase was observed (Fig. 4B). NR5A1-ADSC co-cultured with VEC showed the same results, and no significant difference in corticosterone concentration was observed with ACTH or forskolin stimulation (Fig. 4B). In all cases, the ACTH or forskolin stimulation of Ctrl-ADSC or co-culture of Ctrl-ADSC and VEC resulted in corticosterone levels in the culture medium below the limit of detection. From the above results, the expression of Mc2r and Mrap increased at the mRNA level when co-cultured with VEC, but an increase in corticosterone production in response to ACTH stimulation was not detected.

Co-culture with either lymphatic endothelial cells or ADSCs did not affect corticosterone production by NR5A1-ADSCs, and cell-to-cell adhesion was required for VECs to increase corticosterone production in NR5A1-ADSCs

To determine whether the corticosterone-increasing effect is specific to VECs, NR5A1-ADSCs were co-cultured with lymphatic endothelial cells, another type of endothelial cell or ADSCs to examine steroid production (Fig. 5A). ADSCs served as a source of differentiated steroid-producing cells, and in addition to their stem cell properties, ADSCs produce trophic factors and harbors cytoprotective properties.

Figure 5
figure 5

Co-culture with LECs or ADSCs did not affect corticosterone and testosterone production. (A) NR5A1-ADSCs (5.0 × 104 cells/well) were co-cultured with 0, 5.0 × 103, 2.5 × 104, 5.0 × 104 of LECs or ADSCs. Corticosterone, testosterone, and corticosterone-testosterone ratio in media co-cultured with LECs (B) or ADSCs (C). D, NR5A1-ADSCs (5.0 × 104 cells/well), and VECs (5.0 × 104 cells/well) were cultured in the upper insert and bottom dish of the transwell culture system, respectively. (E) corticosterone, and testosterone levels were measured using ELISA, and the B/T ratio was calculated. Statistical analysis was performed using a one-way ANOVA. ns; not significant. ADSCs, adipose-derived stem cells; ANOVA, analysis of variance; B/T, corticosterone to testosterone ratio; ELISA, enzyme-linked immunosorbent assay; LEC, lymphatic endothelial cells; NR5A1, Nuclear receptor subfamily 5 group A member 1; VECs, vascular endothelial cells.

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When NR5A1-ADSCs were co-cultured with LECs or ADSCs instead of VECs, neither LECs nor ADSCs affected corticosterone and testosterone production by NR5A1-ADSCs (Fig. 5B,C). These results suggest that the enhancement of corticosterone is specific to the vascular endothelium.

The mechanisms underlying the corticosterone-increasing effects of VECs may involve the activation of signaling via cell–cell contact, secretion factors from VECs, or a combination of both. To analyze the impact of VEC secretion factors, NR5A1-ADSCs and VECs were cultured separately using a transwell culture system, with NR5A1-ADSCs cultured in the lower well and VECs cultured in the upper insert well. The enzyme-linked immunosorbent assay (ELISA) revealed that neither corticosterone nor testosterone production increased in NR5A1-ADSCs co-cultured with VECs using the transwell system (Fig. 5D,E).

Extracellular matrix increased corticosterone production in NR5A1-induced steroidogenic cells

Increased corticosterone production by co-culture with VECs was not detected in transwell cultures, suggesting that the extracellular matrix and matricellular proteins of VECs are more important than the liquid factors and extracellular vesicles secreted from VECs. LECs, similar to VECs, are endothelial cells comprising the vascular endothelium but do not promote corticosterone production (Fig. 5B). Therefore, gene expression levels of VECs and LECs were compared to identify factors predominantly expressed in VECs. We utilized publicly available datasets, GSE2203432 and GSE2622933, to compare gene expression profiles. Specifically, we focused on adhesion factors, extracellular matrices, and plasma membrane proteins that exhibit high expression in VECs. Supplementary Fig. S2 displays four genes validated by real-time PCR that are highly expressed in VECs and mouse adrenal cortex. However, inhibiting their expression in VECs using siRNA did not reduce corticosterone production in NR5A1-ADSCs.

In the public datasets, the basement membrane components laminin alpha 3 (Lama3), laminin alpha 5 (Lama5), laminin B1 (lamb1), collagen type IV, alpha 1, 2, 3, and 4 (Col4a1, 2, 3, and 4), and collagen, type XV, alpha1 (Col15a1) were highly expressed in VECs compared to LECs. Thus, we focused on type IV collagen and laminin. NR5A1-ADSCs were cultured on type IV collagen- and laminin-coated plates and the secretion of corticosterone and testosterone was examined using ELISA (Fig. 6A). Corticosterone production of NR5A1-ADSCs cultured in plates coated with type IV collagen or laminin alone showed an increasing trend compared to uncoated plates, although no significant differences were observed. Corticosterone production of ADSCs cultured in plates coated with type IV collagen and laminin was significantly increased, whereas testosterone production remained unchanged (Fig. 6B).

Figure 6
figure 6

Corticosterone secretion from NR5A1-ADSCs was increased by culture in collagen and laminin-coated dish. (A) ADSCs were inoculated with adeno-NR5A1, cultured overnight, plated in type IV collagen, laminin, or type IV collagen and laminin-coated plate, and cultured for 6 days. (B) The culture medium was collected, and the corticosterone and testosterone levels were measured using ELISA. Statistical analysis was performed using a one-way ANOVA (*p < 0.05, ns; not significant). ADSCs, adipose-derived stem cells; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; NR5A1, nuclear receptor subfamily 5 group A member 1.

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Discussion

Our results revealed that co-culture with VECs did not alter testosterone production in NR5A1-induced steroidogenic cells generated from mouse ADSCs. However, it increased the production of corticosterone, the primary glucocorticoid in mice. Furthermore, co-culture with VEC induced the expression of Mc2r and Mrap in NR5A1-ADSCs at the mRNA level. Although NR5A1-ADSCs co-cultured with VEC were expected to be ACTH-responsive, no increase in corticosterone secretion was detected in response to ACTH stimulation. This is probably owing to insufficient induction of Mc2r expression, and the molecular mechanism by which NR5A1 cannot induce high expression of Mc2r in mouse ADSCs is unknown.

The adrenal cortex is a highly vascularized tissue, and steroid hormones produced within the adrenal cortex are transported through the bloodstream23. During adrenal development, the development and differentiation of steroid-producing cells interact with the development of the vascular network27. Recent single-cell analysis of mouse embryos has revealed the presence of a cell population expressing cadherin 5- and CD31-positive vascular cells within the adrenal primordium as early as embryonic day 9 (E9.0)34. This suggests that VECs may play a role during the early stages of adrenal and gonadal development. Conversely, steroid-producing cells secrete angiogenic factors such as VEGF and EG-VEGF, which are required for vascular endothelial growth and sprouting35. Notably, ACTH stimulation enhances VEGFA production in adrenocortical cells, and VEGFA expression is mediated by NR5A125,36,37,38. NR5A1-ADSCs showed enhanced VEGFA production, suggesting that ADSC-derived steroidogenic cells have the potential to produce VEGFA in an NR5A1-dependent manner.

VECs are also important in the development of several tissues, as paracrine factors secreted by endothelial cells stimulate the differentiation of neighboring cells39,40. In addition to secreted factors, the extracellular matrix of endothelial cells also promotes cell proliferation and differentiation41. Using these actions of endothelial cells, the tissues and cells co-transplanted with endothelial cells show enhanced viability42,43,44. Cytoprotective effects have been observed in endothelial and mesenchymal stem cells that secrete trophic factors. When NR5A1-induced steroidogenic cells and VECs were cultured in the transwells, no corticosterone-increasing effects by vascular endothelium were detected, suggesting that the endothelial cell effects are not owing to secreted factors, suggesting that cell-to-cell adhesion is required. Other endothelial cells, such as lymphatic endothelium or ADSCs, did not show the same corticosterone-increasing effects as vascular endothelium, highlighting their specificity for VECs. From publicly available data, we identified genes expressed in both the adrenal cortex and VECs. Specifically, Exoc3l2, Sparcl1, Entpd1, and Pcdh17 were identified as genes predominantly expressed in VECs, with notable expression levels also observed in the mouse adrenal cortex. However, the role of these genes in the adrenal cortex remains poorly understood. Inhibition of Exoc3l2, Sparcl1, and Entpd1 expression in VECs resulted in reduced Star expression in NR5A1-induced steroidogenic cells but did not affect corticosterone production.

The extracellular matrix (ECM) is currently considered an important component of the adrenocortical microenvironment, playing a key role in the regeneration and maintenance of the three zones of the adrenal cortex45. ECM-derived fibronectin, laminin, and collagen IV promote the proliferation of human fetal adrenal cells. Furthermore, fibronectin and laminin had an inhibitory effect on ACTH-induced cortisol production, whereas collagen IV had a promotive effect46,47. Results from investigations using coated dishes showed that collagen and laminin, which are ECM components of the vascular endothelium, promoted NR5A1-ADSC corticosterone production.

NR5A1-induced steroidogenic cells derived from mesenchymal cells produce both adrenal and gonadal steroids5,7,8,18,19. To achieve steroid replacement by cell therapy for adrenocortical insufficiency, cells with more adrenocortical properties are required; NR5A1-induced steroidogenic cells co-cultured with VECs become more adrenocortical through the action of an ECM. Although the molecular mechanism of the corticoid-increasing effects of VECs remains unclear, these findings may have potential implications in cellular therapies aimed at steroid replacement for adrenocortical insufficiency.

Methods

Cell cultures

The animal experimental protocols were approved by the committee of Fukuoka University (approval number: 2214035) and adhered to the ARRIVE guidelines and American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. The mice were euthanized using 5% isoflurane inhalation anesthesia. As described previously18,48, for the preparation of ADSCs, subcutaneous adipose tissue was extracted from 10-week-old male C57BL/6 J mice (The Jackson Laboratory, Yokohama, Japan) and digested in 0.2% collagenase at 37ºC for 30 min, and centrifuged at 400 × g for 10 min. The precipitated stromal vascular fraction was cultured for 2 weeks in α-minimum essential medium containing 20% horse serum (Thermo Fisher Scientific, Waltham, MA, USA) in a 5% CO2 incubator at 37 °C, and adherent cells were used in experiments up to passage 618,48. Primary cultures of C57BL/6-derived VECs and LECs (Cell Biologics, Chicago, IL, USA) were cultured on 0.1% gelatin-coated culture dishes using the EGM2-endothelial cell growth medium-2 bullet kit (Lonza, Basel, Switzerland). VECs up to passage 8 were used in experiments.

Recombinant adenoviral vectors and inoculation

The genetic recombination experiment was approved by the Fukuoka University Safety Committee for Genetic Recombination Experiments (No. 504). A recombinant adenoviral expression vector containing human NR5A1 cDNA or LacZ cDNA was prepared using the Adeno-X Adenoviral system 3 (CMV, Red) (Takara Bio, Shiga, Japan)19. For the infection, ADSCs were cultured overnight in DMEM containing 10% FBS (Cytiva, Marlborough, MA, USA) and adenovirus solution (multiplicity of infection [MOI] = 25), then subsequently washed with a fresh medium. ADSCs infected with adeno-NR5A1 were named NR5A1-ADSCs, whereas those with adeno-lacZ were designated Ctrl-ADSCs.

Detection of expression of NR5A1 in NR5A1-ADSCs

ADSCs on day 3 after adenovirus infection were subjected to immunocytochemistry and western blotting. For immunocytochemistry, NR5A1-ADSCs were fixed in 4% paraformaldehyde (PFA) for 20 min and incubated in a blocking buffer for 30 min. After washing with PBS, the cells were incubated overnight in an anti-STF1 antibody (Cell Signaling Technology, Danvers, MA, USA) solution at 4 °C and in a secondary antibody solution containing Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoReasearch, West Grove, PA, USA). For western blot analysis, NR5A1-ADSCs were lysed in RIPA buffer, centrifuged (10,000 × g, 4 °C, 10 min), and cell supernatant was obtained. After the protein assay, each cell supernatant was expanded using SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was subjected to antibody reaction with anti-STF-1 antibody or anti-HSP90 (Cell Signaling Technology) at 4 °C overnight. Membranes were washed with TBS-0.05% Tween 20 and incubated with a secondary antibody for 2 h at room temperature, and NR5A1 expression was visualized using ECL detection reagents (Cytiva) and an image analyzer 680 (Cytiva). The expression of endogenous control was detected in HSP90. The intensity of the NR5A1 and HSP90 bands was measured using the ImageQuant TL ver. 8.1 (https://www.cytivalifesciences.com/en/us/shop/protein-analysis/molecular-imaging-for-proteins/imaging-software/imagequant-tl-10-2-analysis-software-p-28619)(Cytiva).

Histological analysis

Adrenal glands were removed from 10 to 14-week-old C57BL/6 J mice following euthanasia under anesthesia. The tissues were fixed in 4% PFA in PBS and subsequently embedded in paraffin. Immunohistochemistry was performed using primary antibody against NR5A1 (STF1) (Cell Signaling Technology). Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoReasearch) was used as a secondary antibody. Images were acquired using a BZ-710 fluorescent microscope (KEYENCE, Kyoto, Japan). Lycopersicon Esculentum (Tomato) Lectin-DyLight 649 (Vector Laboratories, Burlingame, CA, USA) was used to visualize the vascular endothelium.

Co-culture of NR5A1-ADSCs with VECs and cell sorting

ADSCs were seeded in 6-well plates (5.0 × 104 cells/well), cultured overnight in DMEM containing 10% FBS, and subsequently inoculated with adeno-NR5A1 or adeno-lacZ (MOI = 25). Three days after inoculation, VECs (0.5 × 104, 2.5 × 104, or 5.0 × 104 cells) were added to the NR5A1-ADSCs or Ctrl-ADSCs, then co-cultured for 4 days in DMEM containing 10% FBS. To isolate adenovirus-inoculated cells from the co-culture, DsRed-positive cells were sorted using a FACSAria Fusion (BD Biosciences, San Jose, CA, USA). For the transwell culture, NR5A1-ADSCs were seeded on the lower dish, and VECs were seeded on the upper insert (membrane pore size; 0.8 µm). In the ACTH or forskolin stimulation assay, 2 × 104 ADSCs were seeded in a 12-well plate and cultured overnight the day before adenovirus infection. Adeno-NR5A1 or adeno-lacZ was infected at a MOI of 25 (day 0). On day 3 after infection, 2 × 104 VECs were added and cultured, and on day 5 after infection, the medium (DMEM 10% FBS) was replaced with a medium containing 2 µM ACTH (Merck, Darmstadt, Germany) or 10 µM forskolin (Merck), and the cells were cultured until day 7 after infection. The concentration of corticosterone in the medium was measured by ELISA.

Quantitative RT-PCR

Total RNA was extracted using a Fast Gene RNA Basic Kit (Nippon Genetics, Tokyo, Japan), followed by cDNA synthesis using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). Quantitative RT-PCR (qPCR) was performed using TB Green Premix Ex Taq™ II (Takara Bio) and a LightCycler96 (Roche Diagnostics, Basel, Switzerland). Beta-actin (Actb) was used as an internal reference gene for normalization. Primer sequences for qPCR experiments are listed in Supplementary Table S1.

Steroid hormone and VEGFA measurement

Corticosterone and testosterone levels in the culture medium were measured using ELISA kits (Cayman Chemical, Ann Arbor, MI, US). The detection limits for corticosterone and testosterone were 8.2 and 3.9 pg/mL, respectively. Cortisol and aldosterone were measured using LC–MS/MS (ASKA Pharma Medical, Kanagawa, Japan). The detection limits for cortisol, and aldosterone were 50, and 5 pg/mL, respectively. VEGFA concentration was measured using a mouse VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Gene silencing by siRNA

Exoc3l2, Sparcl1, and Entpd1 expression was suppressed using siRNAs designed using siDirect ver.2 (http://sidirect2.rnai.jp/). The siRNA sequences for each target gene are listed in Supplementary Table S2. For transfection, VECs were seeded into 12-well plates (3.0 × 104 cells/well) and transfected with 10 pmol of target or non-target siRNAs (MISSION siRNA universal control#1, Merck) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) following the manufacturer’s instructions.

Culture in collagen or laminin-coated dishes

For cell culture in coated dishes, the 24-well plates were coated with recombinant type IV collagen (Merck) and Laminin (Merck) at 4 µg/cm2 for 2 h at 37 °C, then subsequently washed with PBS. Uncoated, collagen IV-, laminin-, or collagen IV and laminin-coated plates were seeded with NR5A1-ADSCs 1 day after infection with adeno-NR5A1 before being cultured in DMEM containing 10% FBS. The medium was replaced on days 3 and 5 following the adenovirus infection, and the culture supernatant was collected on day 7.

Statistical analysis

Statistical analysis was performed using GraphPad Prism ver. 9 (https://www.graphpad.com/features) (GraphPad Software, San Diego, CA, USA). All data are presented as the means ± standard error. Comparative analysis was performed using the Student’s t-test and one-way analysis of variance (ANOVA). Tukey, Dunnett, and Sidak’s post-hoc analyses were performed for multiple comparisons. Significance was determined at a threshold of p < 0.05.