RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue

Background: RNase A (the bovine equivalent to human RNase 1) and RNase 5 (angiogenin) are two closely related ribonucleases. RNase 5 is described as a powerful angiogenic factor. Whether RNase A shares the same angiogenic characteristic, or interferes with vessel growth as demonstrated for arteriogenesis, has never been investigated and is the topic of this present study. Methods and Results: To investigate whether RNase A shows a pro‐ or anti-angiogenic effect, we employed a murine hindlimb model, in which femoral artery ligation (FAL) results in arteriogenesis in the upper leg, and, due to provoked ischemia, in angiogenesis in the lower leg. C57BL/6J male mice underwent unilateral FAL, whereas the contralateral leg was sham operated. Two and seven days after the surgery and intravenous injection of RNase A (50 μg/kg dissolved in saline) or saline (control), the gastrocnemius muscles of mice were isolated from the lower legs for (immuno-) histological analyses. Hematoxylin and Eosin staining evidenced that RNase A treatment resulted in a higher degree of ischemic tissue damage. This was, however, associated with reduced angiogenesis, as evidenced by a reduced capillary/muscle fiber ratio. Moreover, RNase A treatment was associated with a significant reduction in leukocyte infiltration as shown by CD45+ (pan-leukocyte marker), Ly6G+ or MPO+ (neutrophils), MPO+/CitH3+ [neutrophil extracellular traps (NETs)], and CD68+ (macrophages) staining. CD68/MRC1 double staining revealed that RNase A treated mice showed a reduced percentage of M1-like polarized (CD68+/MRC1−) macrophages whereas the percentage of M2-like polarized (CD68+/MRC1+) macrophages was increased. Conclusion: In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.</p

Relevance of lymphocytes in collateral artery growth (arteriogenesis)

Arteriogensis is a natural bypass growth of preexisting arteriole to fully functioning arteries that rescue organs from ischemic damage and necrosis. Neutrophil extravasation and mast cell degranulation are the early cellular events in this process. The Role of B cells in arteriogenesis has never been studied. There is contradictory evidence of CD4+ and CD8+ T cells role in arteriogenesis. My study aims to identify the role of lymphocytes in arteriogenesis. Using Rag1 KO mice, I found lack of B and T cells Rag1 KO mice showed impaired arteriogenesis. Macrophage phenotype analysis in perivascular space revealed that impaired arteriogenic response in Rag1 KO mice was associated with increased number of inflammatory (CD68+MRC1-) macrophages and reduced number of regenerative (CD68+MRC1+) macrophages indicating the role of adaptive immune system in resolving the inflammation and facilitate the collateral artery growth. Next, in WT mice, CD20 mediated B cell depletion resulted in impaired arteriogenesis associated with the reduced regenerative macrophage accumulation. Not only B cells, but fraction of CD20+ T cells and eosinophils were also depleted by CD20 depletion. Flow cytometry analysis showed that CD20 expression was not limited to B cells but also other cell types, i.e., CD4 T cells, CD8 T cells, γδ T cells, and eosinophils. Imaging cytometry using two clones of CD20 antibodies confirms this observation and explains why CD20 depletion affected CD4 T cells, γδ T cells, and eosinophils. These results draw a tentative conclusion on the role of B cells in arteriogenesis. B cell-deficient (JHT KO) mice provide a better alternative to study the role of B cells in arteriogenesis. B cell-deficient mice showed reduced arteriogenesis; however, inflammatory (CD68+MRC1-) and regenerative (CD68+MRC1+) macrophage accumulation were not affected by B cells absence. Histology immunofluorescence analysis showed the presence of CD20+ cells in B cell-deficient mice, and these cells might be the reason why B cell-deficient mice have similar macrophages compared to WT mice. CD20 depletion in B cell-deficient mice showed a strong negative effect on arteriogenesis, associated with an increased number of inflammatory (CD68+MRC1-) macrophages and decreased regenerative (CD68+MRC1+) macrophages. Next, analysis from TCRα KO mice showed me that lack of CD4 and CD8 T cells does not impact arteriogenesis and favored regenerative (CD68+MRC1+) macrophage accumulation. Nevertheless, surprisingly, γδ T cell depletion negatively affected arteriogenesis in TCRα KO mice and WT mice. Furthermore, γδ T cell depletion also reduced the regenerative (CD68+MRC1+) macrophage accumulation. My study, for the first time, identified the involvement of γδ T cell in arteriogenesis. γδ T cells expressing IFN-γ in arteriogenesis. Macrophage activation status analysis showed that CD169 (Siglec-1) expression decreased in γδ T cell depleted mice. CD169 macrophages showed vascular growth-promoting properties as analyzed by expression of IL-10 and PDGFβ. In conclusion, my study provides convincing evidence of the contribution of B cells and γδ T cells in collateral artery growth (arteriogenesis)

Rag1 Deficiency Impairs Arteriogenesis in Mice

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

Association of GSTT1 and GSTM1 polymorphisms in South Indian Epilepsy Patients

783-787Experimental studies suggest that oxidative stress is one of the contributing factors in the onset of epileptic seizures. Glutathione S-transferases (GSTs) are able to conjugate electrophilic compounds, and thus possess neuroprotective role by removing exogenous and endogenous oxidants, detoxifying therapeutic drugs, environmental toxins through conjugation with glutathione (GSH). Several studies from different ethnic groups showed that polymorphisms of the GST gene have been associated with Epilepsy. In the present study, we investigated the association of GST polymorphism in the South Indian epilepsy patients population. A total 371 samples (110 cases and 261 controls) were genotyped for the GST1 and GSTM1 polymorphism by multiplex PCR method. We observed a significant association of GSTT1 null polymorphism in patients with epilepsy. The frequency of the GSTT1 null genotype was found to be significantly higher in cases (35.45 %) than the controls (18.39 %) (OR: 2.44, 95%CI: 1.4-4.02, P <0.0001). In contrast, the frequency of the GSTM1 null variant was significantly lower in cases (11.81%) than controls (32.95%) (OR: 0.27, 95%CI: 0.14-0.51, P <0.001) indicating a protective role. These results indicated that individuals who have GSTT1 null variant are at higher risk for developing seizure than those of GSTT1 wild genotype. On the other hand, individuals carrying GSTM1 null variant showed protective role against seizure. Further, these two null variants did not show any significant association with antiepileptic drug-induced skin rash

Depletion of γδ T Cells Leads to Reduced Angiogenesis and Increased Infiltration of Inflammatory M1-like Macrophages in Ischemic Muscle Tissue

γδ T cells, a small subset of T cells in blood, play a substantial role in influencing immunoregulatory and inflammatory processes. The functional impact of γδ T cells on angiogenesis in ischemic muscle tissue has never been reported and is the topic of the present work. Femoral artery ligation (FAL) was used to induce angiogenesis in the lower leg of γδ T cell depleted mice and wildtype and isotype antibody-treated control groups. Gastrocnemius muscle tissue was harvested 3 and 7 days after FAL and assessed using (immuno-)histological analyses. Hematoxylin and Eosin staining showed an increased area of tissue damage in γδ T cell depleted mice 7 days after FAL. Impaired angiogenesis was demonstrated by lower capillary to muscle fiber ratio and decreased number of proliferating endothelial cells (CD31+/BrdU+). γδ T cell depleted mice showed an increased number of total leukocytes (CD45+), neutrophils (MPO+) and neutrophil extracellular traps (NETs) (MPO+/CitH3+), without changes in the neutrophils to NETs ratio. Moreover, the depletion resulted in a higher macrophage count (DAPI/CD68+) caused by an increase in inflammatory M1-like macrophages (CD68+/MRC1−). Altogether, we show that depletion of γδ T cells leads to increased accumulation of leukocytes and M1-like macrophages, along with impaired angiogenesis

Contribution of the Potassium Channels KV1.3 and KCa3.1 to Smooth Muscle Cell Proliferation in Growing Collateral Arteries

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 KV1.3 and KCa3.1 for SMC proliferation in arteriogenesis. Employing a murine hindlimb model of arteriogenesis, we found that blocking KV1.3 with PAP-1 or KCa3.1. 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 &alpha; smooth muscle-actin (&alpha;SM-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 KV1.3. blockade, but not when KCa3.1 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 KV1.3 but not KCa3.1 contributes to SMC proliferation in arteriogenesis

C3 Deficiency Leads to Increased Angiogenesis and Elevated Pro-Angiogenic Leukocyte Recruitment in Ischemic Muscle Tissue

The complement system is a potent inflammatory trigger, activator, and chemoattractant for leukocytes, which play a crucial role in promoting angiogenesis. However, little information is available about the influence of the complement system on angiogenesis in ischemic muscle tissue. To address this topic and analyze the impact of the complement system on angiogenesis, we induced muscle ischemia in complement factor C3 deficient (C3−/−) and wildtype control mice by femoral artery ligation (FAL). At 24 h and 7 days after FAL, we isolated the ischemic gastrocnemius muscles and investigated them by means of (immuno-)histological analyses. C3−/− mice showed elevated ischemic damage 7 days after FAL, as evidenced by H&amp;E staining. In addition, angiogenesis was increased in C3−/− mice, as demonstrated by increased capillary/muscle fiber ratio and increased proliferating endothelial cells (CD31+/BrdU+). Moreover, our results showed that the total number of leukocytes (CD45+) was increased in C3−/− mice, which was based on an increased number of neutrophils (MPO+), neutrophil extracellular trap formation (MPO+/CitH3+), and macrophages (CD68+) displaying a shift toward an anti-inflammatory and pro-angiogenic M2-like polarized phenotype (CD68+/MRC1+). In summary, we show that the deficiency of complement factor C3 increased neutrophil and M2-like polarized macrophage accumulation in ischemic muscle tissue, contributing to angiogenesis

Modulation of Rxr&alpha; Expression in Mononuclear Phagocytes Impacts on Cardiac Remodeling after Ischemia-Reperfusion Injury

Retinoid X receptors (RXRs), as members of the steroid/thyroid hormone superfamily of nuclear receptors, are crucial regulators of immune response during health and disease. RXR subtype expression is dependent on tissue and cell type, RXR&alpha; being the relevant isoform in monocytes and macrophages. Previous studies have assessed different functions of RXRs and positive implications of RXR agonists on outcomes after ischemic injuries have been described. However, the impact of a reduced Rxr&alpha; expression in mononuclear phagocytes on cardiac remodeling after myocardial infarction (MI) has not been investigated to date. Here, we use a temporally controlled deletion of Rxr&alpha; in monocytes and macrophages to determine its role in ischemia-reperfusion injury. We show that reduced expression of Rxr&alpha; in mononuclear phagocytes leads to a decreased phagocytic activity and an accumulation of apoptotic cells in the myocardium, reduces angiogenesis and cardiac macrophage proliferation in the infarct border zone/infarct area, and has an impact on monocyte/macrophage subset composition. These changes are associated with a greater myocardial defect 30 days after ischemia/reperfusion injury. Overall, the reduction of Rxr&alpha; levels in monocytes and macrophages negatively impacts cardiac remodeling after myocardial infarction. Thus, RXR&alpha; might represent a therapeutic target to regulate the immune response after MI in order to improve cardiac remodeling

c.*84G&#62;A mutation in CETP is associated with coronary artery disease in South Indians

Background: Coronary Artery Disease (CAD) is one of the leading causes of mortality worldwide. It is a multi-factorial disease and several studies have demonstrated that the genetic factors play a major role in CAD. Although variations in Cholesteryl Ester Transfer Protein (CETP) gene are reported to be associated with CAD, this gene has not been studied in South Indian populations. Hence we evaluated the CETP gene variations in CAD patients of South Indian origin. Methods: We sequenced all the exons, exon-intron boundaries and UTRs of CETP in 323 CAD patients along with 300 ethnically and age matched controls. Variations observed in CETP were subjected to various statistical analyses. Results and Discussion: Our analysis revealed a total of 13 variations. Of these, one 3’UTR variant rs1801706 (c.*84G&#62;A) was significantly associated with CAD (genotype association test: OR = 2.16, 95% CI: 1.50–3.10, p = 1.88x10-5 and allelic association test: OR = 1.92, 95% CI: 1.40–2.63, p = 2.57x10-5 ). Mutant allele “A” was observed to influence the higher concentration of mRNA (p = 7.09×10-3, R2 = 0.029 and &#946; = 0.2163). Since expression of CETP has been shown to be positively correlated with the risk of CAD, higher frequency of “A” allele (patients: 22.69% vs. controls: 13%) reveals that c.*84G&#62;A is a risk factor for CAD in South Indians. Conclusions: This is the first report of the CETP gene among South Indians CAD patients. Our results suggest that rs1801706 (c.*84G&#62;A) is a risk factor for CAD in South Indian population