Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection. To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of stromal cells from murine lung tissue Basic Protocol 2: Flow cytometry assessment of lung stromal populations Basic Protocol 3: Purification of lung fibroblastic stromal cells Alternate Protocol: Positive selection of fibroblastic stromal cells.
© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Diabetes is associated with a significantly elevated risk of heart failure. However, despite extensive efforts to characterize the phenotype of the diabetic heart, the molecular and cellular protagonists that underpin cardiac pathological remodeling in diabetes remain unclear, with a notable paucity of data regarding the impact of diabetes on non-myocytes within the heart. Here we aimed to define key differences in cardiac non-myocytes between spontaneously type-2 diabetic (db/db) and healthy control (db/h) mouse hearts. Single-cell transcriptomic analysis revealed a concerted diabetes-induced cellular response contributing to cardiac remodeling. These included cell-specific activation of gene programs relating to fibroblast hyperplasia and cell migration, and dysregulation of pathways involving vascular homeostasis and protein folding. This work offers a new perspective for understanding the cellular mediators of diabetes-induced cardiac pathology, and pathways that may be targeted to address the cardiac complications associated with diabetes.
© 2023 The Authors.

It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway.
An implant was inserted into the right tibia of 16-week-old female C57BL/6 mice (n = 38). Mice received anti-VEGF receptor-1 (VEGFR-1) antibody (25 mg/kg) and VEGF receptor-2 (VEGFR-2) antibody (25 mg/kg; n = 19) or an isotype control antibody (n = 19). Flow cytometric (n = 4/group) and immunofluorescent (n = 3/group) analyses were performed at two weeks post-implantation to detect the distribution and density of CD31hiEMCNhi endothelium. RNA sequencing analysis was performed using sorted CD31hiEMCNhi endothelial cells (n = 2/group). Osteoblast lineage cells expressing osterix (OSX) and osteopontin (OPN) were also detected with immunofluorescence. Mechanical pull-out testing (n = 12/group) was used at four weeks post-implantation to determine the strength of the bone-implant interface. After pull-out testing, the tissue attached to the implant surface was harvested. Whole mount immunofluorescent staining of OSX and OPN was performed to determine the amount of osteoblast lineage cells.
Flow cytometry revealed that anti-VEGFR treatment decreased CD31hiEMCNhi vascular endothelium in the peri-implant bone versus controls at two weeks post-implantation. This was confirmed by the decrease of CD31 and endomucin (EMCN) double-positive cells detected with immunofluorescence. In addition, treated mice had more OPN-positive cells in both peri-implant bone and tissue on the implant surface at two weeks and four weeks, respectively. More OSX-positive cells were present in peri-implant bone at two weeks. More importantly, anti-VEGFR treatment decreased the maximum load of pull-out testing compared with the control.
VEGF pathway controls the coupling of angiogenesis and osteogenesis in orthopaedic implant osseointegration by affecting the formation of CD31hiEMCNhi endothelium. Cite this article: Bone Joint J 2019;101-B(7 Supple C):108-114.