Hirotoki Ohkubo

Suppressed recruitment of alternatively activated macrophages reduces TGF-β1 and impairs wound healing in streptozotocin-induced diabetic mice.

BACKGROUND Diabetes mellitus inhibits wound-induced angiogenesis, impairing the wound healing process and leading to the development of chronic wounds. Impaired healing of diabetic wounds is caused by persistent pro-inflammatory macrophages recruited to the granulation tissue; however, little is known about the phenotype of the macrophages involved in diabetic wound healing. The present study was conducted to examine the involvement of macrophages in impaired wound healing using streptozotocin (STZ)-induced diabetic mice. METHODS Full-thickness skin wounds were created on the backs of mice treated with STZ or vehicle. RESULTS Compared with controls, wound healing and angiogenesis were suppressed in STZ-treated mice, with attenuated expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 in wound granulation tissue. STZ-treated mice exhibited enhanced recruitment of classically activated macrophages (M1) expressing inducible nitric oxide synthase (iNOS) and suppressed recruitment of alternatively activated macrophages (M2) expressing transforming growth factor-beta-1 (TGF-β1). Treatment of diabetic mice with TGF-β1 restored wound healing and angiogenesis and normalized M1/M2 macrophage polarization in the granulation tissue. CONCLUSIONS These results suggest that an imbalance of macrophage phenotypes contributes to impaired wound healing in STZ-induced diabetic mice, and treatment with cytokines derived from M2 macrophages may be an effective therapeutic strategy to increase angiogenesis and promote healing of diabetic wounds.

VEGFR1-positive macrophages facilitate liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion injury.

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.

Vascular Endothelial Growth Factor Receptor Type 1 Signaling Prevents Delayed Wound Healing in Diabetes by Attenuating the Production of IL-1β by Recruited Macrophages

The persistence of proinflammatory macrophages, which are recruited to the granulation tissue, impairs the healing of diabetic wounds. Herein, we examined the role of vascular endothelial growth factor receptor type 1 (VEGFR1) signaling in streptozotocin (STZ)-induced diabetic wound healing. Angiogenesis, lymphangiogenesis, and the healing of full-thickness skin wounds were impaired in STZ-treated wild-type (WT) mice compared with vehicle-treated WT mice, with attenuated recruitment of VEGFR1-positive macrophages expressing vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D to the wound granulation tissue. These phenomena were even more prevalent in STZ-treated VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK(-/-) mice). STZ-treated WT mice, but not STZ-treated VEGFR1 TK(-/-) mice, showed accelerated wound healing when treated with placenta growth factor. Compared with that of STZ-treated WT mice, the wound granulation tissue of STZ-treated VEGFR1 TK(-/-) mice contained more VEGFR1-positive cells expressing IL-1β [a classic (M1) activated macrophage marker] and fewer VEGFR1-positive cells expressing the mannose receptor [CD206; an alternatively activated (M2) macrophage marker]. Treatment of STZ-treated VEGFR1 TK(-/-) mice with an IL-1β-neutralizing antibody restored impaired wound healing and angiogenesis/lymphangiogenesis and induced macrophages in the wound granulation tissue to switch to an M2 phenotype. Taken together, these results suggest that VEGFR1 signaling plays a role in regulating the balance between macrophage phenotypes in STZ-induced diabetic wounds, prevents impaired diabetic wound healing, and promotes angiogenesis/lymphangiogenesis.

Thromboxane A(2) receptor signaling promotes liver tissue repair after toxic injury through the enhancement of macrophage recruitment.

It is thought that thromboxane A(2) (TxA(2)) contributes to the progression of inflammation during acute hepatic injury; however, it is still unknown whether TxA(2) is involved in liver repair. The objective of the present study was to examine the role of TxA(2) receptor (TP) signaling in liver injury and repair in response to toxic injury. Carbon tetrachloride (CCl(4)) was used to induce liver injury in TP knockout (TP(-/-)) mice and wild-type (WT) mice. In WT mice, serum levels of alanine aminotransferase (ALT) and the size of the necrotic area peaked at 24 and 48h, respectively, and then declined. In TP(-/-) mice, the changes in ALT levels were similar to WT mice, but liver regeneration was impaired as evidenced by remained elevated levels of hepatic necrosis and by delayed hepatocyte proliferation, which was associated with the reduced expression of growth factors including interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and hepatocyte growth factor (HGF). In TP(-/-) mice, the accumulation of hepatic CD11b(+)/F4/80(+) macrophages in injured livers was attenuated, and the hepatic expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor, the C-C chemokine receptor (CCR2), was reduced compared to WT. Additionally, the application of the TP receptor agonist, U-46619, enhanced the expression of MCP-1/CCL2 and CCR2 in peritoneal macrophages, which was associated with increased levels of IL-6, TNFα and HGF. These results suggested that TP receptor signaling facilitates liver recovery following CCl(4)-induced hepatotoxicity by affecting the expression of hepatotrophic growth factors, and through the recruitment of macrophages mediated by MCP-1/CCL2-CCR2 expression.

Leukotriene B4 type-1 receptor signaling promotes liver repair after hepatic ischemia/reperfusion injury through the enhancement of macrophage recruitment

Recruited macrophages play a critical role in liver repair after acute liver injury. Leukotriene B4 (LTB4) is a potent chemoattractant for macrophages. In this study, we investigated the role of LTB4 receptor type 1 (BLT1) in liver repair during hepatic ischemia/reperfusion (I/R) injury. BLT1‐knockout mice (BLT1‐/‐) or their wild‐type counterparts (WT) were subjected to partial hepatic I/R. Compared with WT, BLT1‐/‐ exhibited delayed liver repair and hepatocyte proliferation accompanied by a 70% reduction in the recruitment of macrophages and a 70–80% attenuation in hepatic expression of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Disruption of BLT1 signaling also reduced the expression of EGF by 67% on recruited macrophages expressing VEGFR1 in the injured liver. Treatment of WT mice with an EGF‐neutralizing antibody delayed liver repair and reduced macrophage recruitment, compared with control immunoglobulin G (IgG). BLT1 signaling enhanced the expression of VEGF, VEGFR1, and EGF in isolated peritoneal macrophages in vitro. These results indicate that BLT1 signaling plays a role in liver repair after hepatic I/R through enhanced expression of EGF in recruited macrophages and that the development of a specific agonist for BLT1 could be useful for liver recovery from acute liver injury.—Ohkubo, H., Ito, Y., Minamino, T., Mishima, T., Hirata, M., Hosono, K., Shibuya, M., Yokomizo, T., Shimizu, T., Watanabe, M., Majima, M., Leukotriene B4 type‐1 receptor signaling promotes liver repair after hepatic ischemia/reperfusion injury through the enhancement of macrophage recruitment. FASEB J. 27, 3132–3143 (2013). www.fasebj.org

Adhesion of platelets through thromboxane A2 receptor signaling facilitates liver repair during acute chemical-induced hepatotoxicity

AIMS Platelets have been suggested to play an important role in liver regeneration and repair after hepatic resection and acute liver injury. However, the underlying mechanisms of liver repair remain elusive. Signaling through thromboxane prostanoid (TP) receptor participates in inflammation and tissue injury through platelet aggregation. On the other hand, TP receptor signaling also is involved in tissue repair and tumor growth through angiogenesis. The present study was examined whether or not TP receptor signaling contributes to liver repair and sinusoidal restoration from acute liver injury through platelet adhesion to the hepatic sinusoids. MAIN METHODS Carbon tetrachrolide (CCl4) was used to induce acute liver injury in TP receptor knockout mice (TP(-/-) mice) and their wild-type littermates (WT mice). KEY FINDINGS Compared with WT mice, TP(-/-) mice exhibited delayed in liver repair and sinusoidal restoration after CCl4 treatment, which were associated with attenuated hepatic expression of pro-angiogenic factors. Intravital microscopic observation revealed that adhering platelets to the sinusoids was increased in WT livers during the repair phase as compared with TP(-/-) livers, and platelet adhesion was dependent on TP receptor signaling. The levels of hepatocyte growth factor (HGF) in platelets from WT mice treated with CCl4 for 48h were greater than those form TP(-/-) mice, and HGF enhanced the expression of angiogenic factors in cultured human umbilical vein endothelial cells (HUVECs). SIGNIFICANCE These results suggested that TP receptor signaling facilitates liver repair and sinusoidal restoration from acute liver injury through HGF release from platelets adhering to the sinusoids.

BLT1 signalling protects the liver against acetaminophen hepatotoxicity by preventing excessive accumulation of hepatic neutrophils

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils. Signalling of LTB4 receptor type 1 (BLT1) has pro-inflammatory functions through neutrophil recruitment. In this study, we investigated whether BLT1 signalling plays a role in acetaminophen (APAP)-induced liver injury by affecting inflammatory responses including the accumulation of hepatic neutrophils. BLT1-knockout (BLT1−/−) mice and their wild-type (WT) counterparts were subjected to a single APAP overdose (300 mg/kg), and various parameters compared within 24 h after treatment. Compared with WT mice, BLT1−/− mice exhibited exacerbation of APAP-induced liver injury as evidenced by enhancement of alanine aminotransferase level, necrotic area, hepatic neutrophil accumulation, and expression of cytokines and chemokines. WT mice co-treated with APAP and ONO-0457, a specific antagonist for BLT1, displayed amplification of the injury, and similar results to those observed in BLT1−/− mice. Hepatic neutrophils in BLT1−/− mice during APAP hepatotoxicity showed increases in the production of reactive oxygen species and matrix metalloproteinase-9. Administration of isolated BLT1-deficient neutrophils into WT mice aggravated the liver injury elicited by APAP. These results demonstrate that BLT1 signalling dampens the progression of APAP hepatotoxicity through inhibiting an excessive accumulation of activated neutrophils. The development of a specific agonist for BLT1 could be useful for the prevention of APAP hepatotoxicity.

Inhibition of microsomal prostaglandin E synthase-1 facilitates liver repair after hepatic injury in mice

BACKGROUND & AIMS Liver repair following hepatic ischemia/reperfusion (I/R) injury is crucial to survival. This study aims to examine the role of endogenous prostaglandin E2 (PGE2) produced by inducible microsomal PGE synthase-1 (mPGES-1), a terminal enzyme of PGE2 generation, in liver injury and repair following hepatic I/R. METHODS mPGES-1 deficient (Ptges-/-) mice or their wild-type (WT) counterparts were subjected to partial hepatic ischemia followed by reperfusion. The role of E prostanoid receptor 4 (EP4) was then studied using a genetic knockout model and a selective antagonist. RESULTS Compared with WT mice, Ptges-/- mice exhibited reductions in alanine aminotransferase (ALT), necrotic area, neutrophil infiltration, chemokines, and proinflammatory cytokine levels. Ptges-/- mice also showed promoted liver repair and increased Ly6Clow macrophages (Ly6Clow/CD11bhigh/F4/80high-cells) with expression of anti-inflammatory and reparative genes, while WT mice exhibited delayed liver repair and increased Ly6Chigh macrophages (Ly6Chigh/CD11bhigh/F4/80low-cells) with expression of proinflammatory genes. Bone marrow (BM)-derived mPGES-1-deficient macrophages facilitated liver repair with increases in Ly6Clow macrophages. In vitro, mPGES-1 was expressed in macrophages polarized toward the proinflammatory profile. Mice treated with the mPGES-1 inhibitor Compound III displayed increased liver protection and repair. Hepatic I/R enhanced the hepatic expression of PGE receptor subtype, EP4, in WT mice, which was reduced in Ptges-/- mice. A selective EP4 antagonist and genetic deletion of Ptger4, which codes for EP4, accelerated liver repair. The proinflammatory gene expression was upregulated by stimulation of EP4 agonist in WT macrophages but not in EP4-deficient macrophages. CONCLUSIONS These results indicate that mPGES-1 regulates macrophage polarization as well as liver protection and repair through EP4 signaling during hepatic I/R. Inhibition of mPGES-1 could have therapeutic potential by promoting liver repair after acute liver injury. LAY SUMMARY Hepatic ischemia/reperfusion injury is a serious complication that occurs in liver surgery. Herein, we demonstrated that inducible prostaglandin E2 synthase (mPGES-1), an enzyme involved in synthesizing prostaglandin E2, worsens the injury and delays liver repair through accumulation of proinflammatory macrophages. Inhibition of mPGES-1 offers a potential therapy for both liver protection and repair in hepatic ischemia/reperfusion injury.

Cerebral oximetry for cardiac surgery: a preoperative comparison of device characteristics and pitfalls in interpretation

Regional cerebral oximetry using near-infrared spectroscopy devices is commonly used for detecting cerebral ischemia during cardiopulmonary bypass, and aim to avoid poor cerebral perfusion which may result in perioperative neurological impairment. Today, several devices that can detect cerebral ischemia are commercially available. Although these devices operate on the same measurement principles, their algorithms for detecting and calculating cerebral ischemia are different and no criteria for directly comparing values measured by such different devices exist. From January 2017 to August 2017, 80 adult cardiovascular surgery patients were enrolled in the prospective study. In each patient, preoperative regional cerebral oxygen saturation values were measured by two different devices and their correlations with various preoperative factors were evaluated. Regional cerebral oxygen saturation levels were significantly higher for values of FORE-SIGHT ELITE (CAS Medical Systems, Branford, CT, USA) (F value) than those of the INVOS 5100C (Medtronic, Minneapolis, MN, USA) (I value). Scalp–cortex distance, hemoglobin concentration, and the presence or absence of hemodialysis showed significant correlations with ratios of measured values specific to each device (F/I). An appropriate device should be selected according to preoperative patient characteristics, and factors influencing regional cerebral oxygen saturation values should be considered to ensure the correct interpretation of measured values. This research was conducted with the approval of the ethics committee of our university (approval number: B16–96).

Daily transient discontinuation of extracorporeal LVAD to prevent thromboembolism of mechanical aortic valve prosthesis

Patients with mechanical aortic valves are generally contraindicated for left ventricular assist device (LVAD) insertion because the prosthetic valve often becomes fixed in closed position. A 41-year-old woman with mechanical aortic valve prosthesis experienced sudden chest pain and developed cardiogenic shock. A paracorporeal pulsatile LVAD and a monopivot centrifugal pump as a right VAD (RVAD) were implanted. The mechanical aortic valve was intentionally left in place. Soon after the operation, LVAD support was discontinued daily for few seconds to allow the mechanical aortic valve to open and to avoid thrombus formation. The patient was successfully weaned off RVAD and received anticoagulation therapy with warfarin. On postoperative day 141, she was transferred to a university hospital where a HeartMate II LVAD was implanted, and the aortic valve was successfully replaced with a bioprosthetic valve. The patient is currently awaiting heart transplantation.