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Cerebral pericytes are essential components of the neurovascular unit, contributing to blood-brain barrier integrity, regulation of cerebral blood flow, and communication among vascular, glial, and neuronal cells. Despite their central role in brain homeostasis, the functional and molecular diversity of cerebral pericytes remains poorly understood. This study aimed to characterize pericyte heterogeneity and determine whether pericyte molecular identity is dynamically regulated under physiological and pathological conditions.

Three publicly available single-cell/single-nucleus RNAseq datasets (Vanlandewijck et al., Yao et al., Smith et al.) were reanalysed using Seurat. Cell interactomes were computed using NATMI and CellChat. All animal procedures were approved by the Cantonal Veterinary Office Zurich (ZH039/2024, ZH194/2020, ZH165/2019). RNA hybridization combined with immunohistochemistry was performed using the rnaMUSE platform (arcoris bio). A fine-tuned vesselFM and 3D nnU-Net were applied for iDISCO image segmentation and annotation to investigate spatial distribution of pericyte subtypes. QuPath, Fiji, Imaris, ITK-SNAP, and ParaView were used for image analysis and visualization.

Distinct pericyte marker profiles were identified that map to specific vascular beds and anatomical regions. ACE2, proposed as a brain pericyte-specific marker, was expressed heterogeneously among mouse brain pericytes, allowing categorization into two subtypes. ACE2-high pericytes were located closer to ASMA-positive arteriolar termini, whereas ACE2-low pericytes were concentrated in capillary beds (branch order >=4). Heterogeneous expression of CASQ2, OPN, and Igf2 among pericytes was validated at mRNA and protein level. Pericyte molecular signatures were not static, undergoing substantial remodeling under acute ischemic stress, healthy aging, and across circadian cycles.

Cerebral pericytes display a highly plastic molecular phenotype that adapts and responds to both transient signals and chronic physiological shifts, rather than a fixed, uniform identity. These findings provide a more detailed framework for understanding pericyte diversity and suggest that pericyte state should be considered a dynamic variable in future studies of neurovascular function and disease.

Metastasis is a multistep process in which malignant tumor cells (TCs) disseminate from the primary tumor and colonize distant organs, establishing secondary lesions. During this process, TCs interact with endothelial cells (ECs) to obtain nutrients and growth factors. Gap Junction Intercellular Communication (GJIC) plays a critical role in TC-EC interactions and metastatic progression. RNA-sequencing analysis performed in our laboratory on TCs and ECs isolated from lung metastases identified the upregulation of Gja1 in both cell types. Gja1 encodes Connexin 43 (Cx43), the most widely expressed protein forming gap junctions. This study aims to investigate the contribution of Cx43 to TC–EC communication and metastasis formation.

Cx43 expression was validated in ECs and TCs sorted from lung metastases, as well as in lung and liver metastatic tissues, by qPCR, Western blot, and immunofluorescence (IF). Functional TC–EC GJIC was assessed using Calcein AM assays. To evaluate the role of Cx43 in metastasis, Cx43-knockdown (Cx43-KD) and knockout (Cx43-KO) TCs were generated using shRNA and CRISPR/Cas9 targeting Gja1, with control cells (Cx43-CTR) processed in parallel. Cx43 reduction or deletion was confirmed molecularly. In vitro proliferation and spheroid assays evaluated cell viability. Cx43-CTR, -KD, and -KO cells were intravenously injected into C57BL/6 mice to assess lung metastasis and vascular organization.

Gja1 was upregulated in metastatic TCs and ECs. Cx43 expression and functional TC–EC GJIC were confirmed. Cx43-KD and -KO cells showed normal in vitro growth and spheroid formation, indicating preserved viability. Primary tumor formation was unaffected by Cx43 loss. In contrast, lung metastasis was markedly impaired: Cx43-KD cells formed fewer metastases, whereas Cx43-KO cells failed to generate detectable lesions. Metastases derived from Cx43-KD cells exhibited altered vasculature. These findings indicate that Cx43-mediated TC–EC communication is dispensable for primary growth but essential for metastatic colonization and vascular organization.

Cx43 is a critical regulator of tumor–endothelial communication during lung metastasis. While not required for primary tumor growth, it is essential for efficient metastatic colonization and proper vessel formation, highlighting Cx43 as a controversial and interesting target.

Spontaneous vascularization of critical-size bone grafts is not sufficiently rapid to ensure efficient bone formation. Angiogenic gene and protein therapy suffer from safety issues and limitations. Chemically modified mRNAs (cmRNAs) are a promising strategy for the safe transient expression of transgenes. Here we investigated the efficacy of a cmRNA encoding VEGF-A to promote the coupling of vascularization and bone formation in engineered grafts.

Osteogenic grafts were prepared with human bone marrow mesenchymal cells (BMSC) and calcium phosphate microparticles in fibrin, containing 0.3 (Low), 3 (Med) or 30 (High) µg/ml of a VEGF cmRNA. Vascularization, osteogenic commitment and bone formation were assessed 1, 4 and 8 weeks after ectopic implantation in nude mice. The translational potential of VEGF cmRNA was tested in a critical-size defect in rats.

After 1 week in vivo, Low VEGF cmRNA stimulated vascular invasion and density compared to controls. Interestingly, Med and High doses significantly increased endothelial density in the outer portion of the construct, but impaired blood vessel in-growth. Quantification of Osterix+ human progenitors showed that the Low dose significantly promoted osteogenic commitment of BMSC compared to controls, but again higher VEGF doses failed to do so. After 8 weeks, grafts with Low VEGF developed a full bone organ, with mature bone tissue and bone marrow pockets. In contrast, higher VEGF cmRNA concentrations caused significant bone loss. Based on these results, the Low VEGF cmRNA dose was tested in a femoral critical-size defect model in rats. Angiogenesis was assessed by immunofluorescence staining for endothelial cells, while bone formation was evaluated using both µCT and histological analysis. VEGF cmRNA delivery could significantly improve vascular density and bone tissue formation within the defect area compared to the control condition (non-coding cmRNA).

These data show that a VEGF cmRNA-loaded matrix could promote rapid vascularization and bone formation, with a clear dose-dependency. Interestingly, despite the transient nature of gene expression afforded by cmRNAs, this mode of VEGF delivery could kick-start angiogenesis, which was sustained until 8 weeks. The combination of safety and efficacy by cmRNAs within a biomaterial holds promise for bone tissue engineering and regenerative medicine in general.