Probes for CCR2

Chronic infusion of Ang II caused increased of leukocyte (CD45+) content in PVAT in both WT and ACKR2-/- mice. This increase was particularly evident for T cell subsets in WT but not in ACKR2-/-. However the number of macrophages (F4/80+CD11b+) was increased in both groups. Interestingly, ACKR2-/- revealed decreased T cell number infiltrating PVAT upon Ang II infusion in comparison to WT. Hypertension was associated with increased CCR1, CCR2 and CCR3 expression in PVAT in both WT and ACKR2-/-. However, ACKR2-/- revealed higher expression of CCR1 and CCR2 but not CCR3 in comparison to WT. Interestingly, increased expression of CCR5 in PVAT upon Ang II infusion in WT was not observed in ACKR2-/-. Hypertension resulted in increased RANTES level in aorta and PVAT to the same extent in both WT and ACKR2-/-. However, MCP-1, MIP-1 alpha and MIP-1 beta level was higher in ACKR2-/- aorta than in WT aorta upon Ang II infusion. ACKR2 expression was lower in PVAT than in aorta. Interestingly, Ang II administration decreased the expression of ACKR2 in aorta but increased ACKR2 level in thoracic PVAT. RNAscope analysis revealed ACKR2 expression in vascular smooth muscle cells (VSMC). Vessels isolated from ACKR2-/- were protected from vascular dysfunction and revealed reduced ROS production in comparison to WT upon Ang II administration. ACKR2-/- mice developed similar extent of blood pressure increase as WT in hypertension.

Microglia, the parenchymal tissue macrophages in the brain, surround amyloid plaques in brains of individuals with Alzheimer's disease (AD) but are ineffective at clearing amyloid to mitigate disease progression. Recent studies in mice indicate that microglia are derived exclusively from primitive yolk sac hematopoiesis and self-renew without contribution from ontogenically distinct monocytes/macrophages of definitive adult hematopoietic origin. Using a genetic fate-mapping approach to label cells of definitive hematopoietic origin throughout life span, we discovered that circulating monocytes contribute 6% of plaque-associated macrophages in aged AD mice. Moreover, peripheral monocytes contributed to a higher fraction of macrophages in the choroid plexus, meninges, and perivascular spaces of aged AD mice versus WT control mice, indicating enrichment at potential sites for entry into the brain parenchyma. Splenectomy, which markedly reduced circulating Ly6Chi monocytes, also reduced abundance of plaque-associated macrophages of definitive hematopoietic origin, resulting in increased amyloid plaque load. Together, these results indicate that peripherally derived monocytes invade the brain parenchyma, targeting amyloid plaques to reduce plaque load.

The sequence of pathological events in feline hypertrophic cardiomyopathy (fHCM) is still largely unknown, although we know that fHCM is characterized by interstitial remodeling in a macrophage-driven pro-inflammatory environment and that myocardial ischemia might contribute to its progression. This study aimed to gain further insights into the structural changes associated with interstitial remodeling in fHCM with special focus on the myocardial microvasculature and the phenotype of the interstitial cells. Twenty-eight hearts (16 hearts with fHCM and 12 without cardiac disease) were evaluated in the current study, with immunohistochemistry, RNA-in situ hybridization, and transmission electron microscopy. Morphometrical evaluations revealed a statistically significant lower microvascular density in fHCM. This was associated with structural alterations in capillaries that go along with a widening of the interstitium due to the accumulation of edema fluid, collagen fibers, and mononuclear cells that also proliferated locally. The interstitial cells were mainly of fibroblastic or vascular phenotype, with a substantial contribution of predominantly resident macrophages. A large proportion expressed CD34 mRNA, which suggests a progenitor cell potential. Our results indicate that microvascular alterations are key events in the pathogenesis of fHCM and that myocardial interstitial cell populations with CD34+ phenotype play a role in the pathogenesis of the disease.

The blood-brain barrier (BBB) consists of endothelial cells, astrocytic end feet, and pericytes to form a barrier that minimizes the entry of circulating proteins and cells into the brain. However, stroke is known to cause significant damage to the BBB, causing the barrier to become permeable, which allows immune cells and other substances to be extravasated into the brain parenchyma. Preservation of the BBB is associated with better ischemic stroke outcomes; therefore, this synopsis summarizes 3 new studies that aim to characterize specific mechanisms of BBB damage and identify potential therapeutic pathways to preserve barrier integrity. CD36 (cluster of differentiation 36) is a glycoprotein expressed by monocytes and macrophages, as well as by endothelial cells. Previous studies have shown that global knockout of CD36 prevents stroke-induced damage, and Kim et al in 2023 published a study in the Journal of Cerebral Blood Flow and Metabolism titled “Endothelial Cell CD36 Mediates Stroke-Induced Brain Injury via BBB Dysfunction and Monocyte Infiltration in Normal and Obese Conditions,” in which they explore the role of CD36 specifically in endothelial cells. Conditional deletion of CD36 in endothelial cells improved stroke outcomes, as indicated by reduced infarct size and hemispheric swelling. Moreover, this deletion improved survival and motor function. Additionally, CD36 deletion in endothelial cells reduced IgG expression in the brain, indicating improved vascular integrity. There was reduced monocyte infiltration into the brain and reduced MCP-1 (monocyte chemoattractant protein-1) and CCR2 (chemokine receptor type 2) expression in the mice with endothelial cell deletion of CD36. This reduced monocyte trafficking persisted even when normalized for infarct size, suggesting that vascular integrity is maintained independent of cell loss. Intriguingly, endothelial cell-specific deletion of CD36 also made mice resistant to developing an obesity phenotype, providing a potential molecular cause for obesity as a stroke risk factor. In the Proceedings of the National Academy for Science in a publication titled “Myeloid-Derived MIF Drives RIPK1-Mediated Cerebromicrovascular Endothelial Cell Death to Exacerbate Ischemic Brain Injury,” Li et al in 2023 describe how macrophage MIF (migration inhibitory factor) exacerbates endothelial cell death and increases BBB permeability after middle cerebral artery occlusion (MCAo). By treating endothelial cells with MIF and subjecting them to oxygen-glucose deprivation followed by reoxygenation, the authors demonstrated that MIF promotes endothelial cell death specifically by activating RIPK1 (receptor-interacting protein kinase 1). Surgical trauma in both mice and humans increases circulating MIF, and the authors use a perioperative ischemic stroke model to see how this surgically induced increase in MIF affects outcomes after distal MCAo. Two-photon imaging showed that perioperative ischemic stroke mice showed increased adhesion of myeloid cells to ischemic microvascular endothelial cells, and RNAscope analysis showed that MIF expression was increased in microglia surrounding endothelial cells in perioperative ischemic stroke mice. Perioperative ischemic stroke mice also exhibited larger infarct volumes and exacerbated BBB damage. The authors then used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) to delete MIF in myeloid cells, which resulted in reduced expression of necrosis markers phosphorylated RIPK1, phosphorylated RIPK3, and CC3 (cleaved caspase 3) in endothelial cells. Mice with myeloid-deleted MIF showed reduced zonulin-1 loss, a marker of endothelial tight junctions, and sensorimotor deficits after distal MCAo. Peripheral blood mononuclear cells from mice with myeloid-deleted MIF were given after MCAo and resulted in smaller infarct volume and reduced IgG extravasation as compared with mice who were given peripheral blood mononuclear cells from wild types. Endothelial cells were cocultured with peripheral blood mononuclear cells from mice with myeloid-deleted MIF and wild types, and zonulin-1 expression was preserved in endothelial cocultured with peripheral blood mononuclear cells from myeloid-deleted MIF mice. Administration of MIF inhibitor before stroke reduced infarct volume, prevented IgG extravasation, preserved tight junction integrity, and prevented endothelial cell death. Collectively, these data show that myeloid-derived MIF is detrimental to BBB integrity after stroke and deleting this source of MIF can improve outcomes through preserving BBB health. Finally, Li et al in 2023 show in ACS Nano in a publication titled “Inducible Pluripotent Stem Cell-Derived Small Extracellular Vesicles Rejuvenate Senescent Blood-Brain Barrier to Protect Against Ischemic Stroke in Aged Mice” that small extracellular vesicles (sEVs) from induced pluripotent stem cells (iPSCs) are able to restore BBB function in old mice by reversing cellular senescence. Mice treated with iPSC-sEVs showed reduced senescence-associated β-galactosidase, p16, p53, p21, and γ-H2AX (histone family member X), all of which are markers associated with cellular senescence. Pretreatment with iPSC-sEVs before MCAo reduced infarct volume in the aged mice, improved neurological score, and reduced sensorimotor deficits, indicating improved stroke outcomes. Moreover, these mice showed decreased leakage of Evans blue dye into the brain parenchyma and preservation of tight junction proteins, indicating that these sEVs preserve BBB integrity after stroke. Mice treated with sEVs showed decreased immune cell infiltration after stroke and attenuated expression of tumor necrosis factor-α, IL (interleukin)-17, IL-6, and IL-1β, Moreover, sEV treatment reduced ischemia-induced apoptosis of oligodendrocytes and neurons. Reversal of the senescent phenotype of the BBB was tested in vitro, by chemically inducing senescence using D-galactose in endothelial cell cultures and subsequent treatment with iPSC-sEVs. sEV treatment reduced senescence markers and prevented loss of tight junction proteins. Oxygen-glucose deprivation was then used to mimic stroke conditions in these cultures, and sEV treatment preserved angiogenic properties of endothelial cells and reduced dextran leakage in a transwell assay. These experiments affirm the in vivo findings that sEV treatment reverses BBB senescence. The 3 studies summarized in this synopsis show 3 different potential pathways that may serve as a target for preserving BBB function after stroke. Deleting endothelial cell CD36, deleting myeloid-derived MIF, or reversing BBB senescence using iPSC-sEVs resulted in improved vascular integrity and overall better stroke outcomes.