(1) Background: Placental immune cells are playing a very important role in a successful placentation and the prevention of pregnancy complications. Macrophages dominate in number and relevance in the maternal and the fetal part of the placenta. The evidence on the polarization state of fetal and maternal macrophages involved in both, healthy and pregnancy-associated diseases, is limited. There is no representative isolation method for the direct comparison of maternal and fetal macrophages so far. (2) Material and Methods: For the isolation of decidual macrophages and Hofbauer cells from term placenta, fresh tissue was mechanically dissected and digested with trypsin and collagenase A. Afterwards cell enrichment was increased by a Percoll gradient. CD68 is represented as pan-macrophage marker, the surface markers CD80 and CD163 were further investigated. (3) Results: The established method revealed a high cell yield and purity of the isolated macrophages and enabled the comparison between decidual macrophages and Hofbauer cells. No significant difference was observed in the percentage of single CD163⁺ cells in the distinct macrophage populations, by using FACS and immunofluorescence staining. A slight increase of CD80⁺ cells could be found in the decidual macrophages. Considering the percentage of CD80⁺CD163⁻ and CD80⁻CD163⁺ cells we could not find differences. Interestingly we found an increased number of double positive cells (CD80⁺CD163⁺) in the decidual macrophage population in comparison to Hofbauer cells. (4) Conclusion: In this study we demonstrate that our established isolation method enables the investigation of decidual macrophages and Hofbauer cells in the placenta. It represents a promising method for direct cell comparison, enzyme independently, and unaffected by magnetic beads, to understand the functional subsets of placental macrophages and to identify therapeutic targets of pregnancy associated diseases.

Collateral artery growth (arteriogenesis) involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Whereas the proliferation of ECs is directly related to shear stress, the driving force for arteriogenesis, little is known about the mechanisms of SMC proliferation. Here we investigated the functional relevance of the potassium channels K_V1.3 and K_Ca3.1 for SMC proliferation in arteriogenesis. Employing a murine hindlimb model of arteriogenesis, we found that blocking K_V1.3 with PAP-1 or K_Ca3.1. with TRAM-34, both interfered with reperfusion recovery after femoral artery ligation as shown by Laser-Doppler Imaging. However, only treatment with PAP-1 resulted in a reduced SMC proliferation. qRT-PCR results revealed an impaired downregulation of α smooth muscle-actin (αSM-actin) and a repressed expression of fibroblast growth factor receptor 1 (Fgfr1) and platelet derived growth factor receptor b (Pdgfrb) in growing collaterals in vivo and in primary murine arterial SMCs in vitro under K_V1.3. blockade, but not when K_Ca3.1 was blocked. Moreover, treatment with PAP-1 impaired the mRNA expression of the cell cycle regulator early growth response-1 (Egr1) in vivo and in vitro. Together, these data indicate that K_V1.3 but not K_Ca3.1 contributes to SMC proliferation in arteriogenesis.

Arteriogenesis is an inflammatory driven mechanism, describing the growth of a natural bypass from pre-existing collateral arteries to compensate for an occluded artery. The complement system component C3 is a potent natural inflammatory activator. Here, we investigated its impact on the process of collateral artery growth using C3-deficient (C3 −/−) and wildtype control mice in a murine hindlimb model of arteriogenesis. Induction of arteriogenesis by unilateral femoral artery ligation resulted in decreased perfusion recovery in C3 −/− mice on day 7 as shown by Laser Doppler imaging. Immunofluorescence staining revealed a reduced vascular cell proliferation in C3 −/− mice. Gene expression analysis displayed a significant reduction in monocyte chemoattractant protein-1 (MCP-1) expression in C3 −/− mice. Interestingly, 3 days after induction of arteriogenesis, the number of macrophages (CD68⁺) recruited to growing collaterals was not affected by C3 deficiency. However, a significant reduction in inflammatory M1-like polarized macrophages (CD68⁺/MRC1⁻) was noted. Forced mast cell activation by Compound 48/80 as well as exogenous MCP-1 application rescued the number of M1-like polarized macrophages along with perfusion recovery in C3 −/− mice. In summary, this study demonstrates that complement C3 influences arteriogenesis by mediating MCP-1 expression, which is essential for the induction and enhancement of sterile inflammation.

Increasing evidence suggests that lymphocytes play distinct roles in inflammation-induced tissue remodeling and tissue damage. Arteriogenesis describes the growth of natural bypasses from pre-existing collateral arteries. This process compensates for the loss of artery function in occlusive arterial diseases. The role of innate immune cells is widely understood in the process of arteriogenesis, whereas the role of lymphocytes remains unclear and is the subject of the present study. To analyze the role of lymphocytes, we induced arteriogenesis in recombination activating gene-1 (Rag1) knockout (KO) mice by unilateral ligation of the femoral artery. The lack of functional lymphocytes in Rag1 KO mice resulted in reduced perfusion recovery as shown by laser Doppler imaging. Additionally, immunofluorescence staining revealed a reduced vascular cell proliferation along with a smaller inner luminal diameter in Rag1 KO mice. The perivascular macrophage polarization around the growing collateral arteries was shifted to more pro-inflammatory M1-like polarized macrophages. Together, these data suggest that lymphocytes are crucial for arteriogenesis by modulating perivascular macrophage polarization.

Background/Objectives: Clopidogrel is a P2Y₁₂ receptor inhibitor commonly used as antiplatelet therapy for patients with cardiovascular occlusive diseases. However, its role in vascular remodeling remains poorly understood. Platelets orchestrate the sterile inflammation in arteriogenesis, an endogenous process to bypass an occluded artery. Therefore, we investigated the impact of P2Y₁₂-inhibition by Clopidogrel on arteriogenesis. Methods: In this study, we utilized a well-established murine hindlimb model of arteriogenesis. To quantify the growth of collateral arteries, we employed laser-Doppler perfusion measurements and immunohistological analysis of growing compared to resting collateral arteries. Additional immunofluorescence and histological stains were conducted to assess immune cell recruitment and activation. Whole-blood flow cytometry was performed to analyze platelet–leukocyte interactions, and complete blood counts were obtained to quantify leukocyte and platelet numbers. Results: The findings of this study demonstrate that Clopidogrel promotes perfusion recovery following the induction of arteriogenesis compared to controls, attributed to elevated levels of proliferating vascular cells. Furthermore, compared to controls, Clopidogrel treatment significantly enhanced platelet-leukocyte interactions, increasing perivascular mast cell recruitment and degranulation, finally resulting in regenerative macrophage accumulation required for collateral artery growth. Conclusions: Clopidogrel treatment boosts arteriogenesis by enhancing the local regenerative inflammation relevant for vascular cell proliferation. Therefore, P2Y₁₂ inhibition may represent a therapeutic option to effectively promote natural bypass growth in patients with cardiovascular occlusive diseases.

Arteriogenesis, the growth of pre-existing arterioles into functional collateral arteries, represents a key adaptive response to severe arterial stenosis. This process is driven by hemodynamic forces and a tightly coordinated inflammatory cascade. Here, we investigated the effects of pharmacological stimulation of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathway using the phosphodiesterase-5 (PDE5) inhibitor Sildenafil on collateral vessel growth in a murine model of femoral artery ligation (FAL). Flow cytometric analyses revealed that Sildenafil treatment significantly enhanced platelet–leukocyte aggregate formation, a prerequisite for the subsequent initiation of a localized perivascular inflammation. Histological and immunofluorescence analyses further demonstrated a marked increase in mast cell recruitment and degranulation at early time points (days 1 and 3 post-FAL). In addition, Sildenafil promoted perivascular macrophage accumulation on days 3 and 7, with a pronounced shift toward an M2-like pro-regenerative polarization state, ultimately resulting in the enhanced proliferation of vascular cells and the enlargement of collateral diameters. Together, these findings identify Sildenafil as a potent enhancer of arteriogenesis through coordinated immune cell activation, stimulating vascular cell proliferation along with positive collateral outward remodeling. Thus, Sildenafil emerges as a promising therapeutic candidate to promote collateral artery growth in cardiovascular occlusive diseases.