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Advanced techniques combining tissue clearing, deep confocal imaging, and image quantification, enable researchers to characterize complex tissue architectures and how trauma or disease impact on their respective components. Here, we detail methodologies to prepare, immunostain, and clear thick sections of mouse bones. We describe their use to visualize and quantitatively analyze marrow blood vessels and their spatial relationship with stromal skeletal stem/progenitor cells. These approaches can help unravel the stromal-vascular interplay in the bone marrow environment in health and disease. ispartof: STAR Protocols status: published

Hematopoietic stem cell (HSC) maintenance throughout life and engraftment after transplantation during anticancer treatment, depend on support from various bone marrow (BM) niche compartments. Here, by studying physiological homing of fetal HSCs, we show the critical requirement of balanced crosstalk within the multi-component BM niche for enabling HSC-lodgment. Transgene-induced over-production of VEGF by skeletal progenitor cells elicited stromal and endothelial hyper-activation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSC seeding, settlement and survival were drastically impaired. Transcriptome-profiling, flow-cytometry and high-resolution imaging indicated alterations in peri-/endothelial characteristics, vascular permeability, and cellular metabolism, associated with a higher oxidative status and increased oxidative stress within the VEGF-enriched BM-environment. Thus, developmental HSC-homing to bone is controlled by local inter-niche communication regulating stromal-vascular integrity and theoxidative and metabolic milieu.Since irradiation of adult mice also induced stromal VEGF-expression and similar osteo-angiogenic niche changes, our findings can contribute to improving stem cell therapy.

Both reductions in atherogenic lipoproteins and increases in high-density lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative potential of low-density lipoprotein (LDL) lowering and HDL raising gene transfer strategies to induce regression of complex murine atherosclerotic lesions was directly compared. Male C57BL/6 LDL receptor (LDLr)−/− mice were fed an atherogenic diet (1.25% cholesterol and 10% coconut oil) to induce advanced atherosclerotic lesions. A baseline group was killed after 6 months and remaining mice were randomized into a control progression (Adnull or saline), an apolipoprotein (apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdA-I/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks with continuation of the atherogenic diet. Gene transfer with AdLDLr decreased non-HDL cholesterol levels persistently by 95% (p < 0.001) compared with baseline. This drastic reduction of non-HDL cholesterol levels induced lesion regression by 28% (p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery at 12 weeks after transfer. Change in lesion size was accompanied by enhanced plaque stability, as evidenced by increased collagen content, reduced lesional macrophage content, a drastic reduction of necrotic core area, and decreased expression of inflammatory genes. Elevated HDL cholesterol following AdA-I transfer increased collagen content in lesions, but did not induce regression. Apo A-I gene transfer on top of AdLDLr transfer resulted in additive effects, particularly on inflammatory gene expression. In conclusion, drastic lipid lowering induced by a powerful gene transfer strategy leads to pronounced regression and stabilization of advanced murine atherosclerosis.