京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18512号 / 医博第3932号 / 新制||医||1006(附属図書館) / 31398 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 前川 平, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Heart failure contributes to nearly 60,000 deaths per year in the USA and is often caused by hypertension and preceded by the development of left ventricular hypertrophy (LVH). LVH is usually accompanied by intensive interstitial and perivascular fibrosis which may contribute to arrhythmogenic sudden cardiac death. Emerging evidence indicates that LV dysfunction in patients and animal models of cardiac hypertrophy is closely associated with perivascular inflammation. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of pressure-overload-induced LVH: transverse aortic constriction (TAC). Early perivascular inflammation was indicated by accumulation of macrophages and T lymphocytes 24 hours post-TAC and which peaked at day 7. Coronary luminal platelet deposition was observed along with macrophages and lymphocytes at day 3. Also, LV protein levels of VEGF and MCP-1 were significantly increased. Consistent with lymphocyte accumulation, cardiac expression of IL-10 mRNA was elevated. Furthermore, circulating platelet-leukocyte aggregates tended to be higher after TAC, compared to sham controls. Platelets have been shown to modulate perivascular inflammation and may facilitate leukocyte recruitment at sites of inflamed endothelium. Therefore, we investigated the impact of thrombocytopenia in the response to TAC. Immunodepletion of platelets decreased early perivascular accumulation of T lymphocytes and IL-10 mRNA expression, and altered subsequent coronary artery remodeling. The contribution of lymphocytes was examined in Rag1-/- mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation which will contribute to long-term LV remodeling. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. To determine the contribution of granule release from inflammatory cells in the development of LVH, we used Unc13dJinx (Jinx) mice, which contain a single point mutation in Unc13d gene resulting in defects in Munc13-4. Munc13-4 is a limiting factor in vesicular priming and fusion during granule secretion. Therefore, Jinx mice have defects in degranulation of platelets, NK cells, cytotoxic T lymphocytes, neutrophils, mast and other cells. With the use of bone marrow transplantation, Jinx chimeric mice were created to determine whether the ability of hematopoietic cells to secrete granule contents affects the development of LVH. Wild-type mice (WT) that were transplanted with WT bone marrow (WT>WT) and WT mice that received Jinx bone marrow (Jinx>WT) developed LVH and a classic fetal reprogramming response early after TAC (7 days), but at later times (5 weeks), Jinx>WT mice failed to sustain the cardiac hypertrophic response observed in WT>WT mice. No difference in cardiac fibrosis was observed at early or late times. Repetitive injection of WT platelets or platelet releasate restored cardiac hypertrophy in Jinx>WT mice. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted, likely from platelets. In conclusion, our studies demonstrate that platelets and lymphocytes are involved in early perivascular inflammation post-TAC, which may contribute to later remodeling in the setting of LVH. Factors released from hematopoietic cells, including platelets, in a Munc13-4-dependent manner are required to promote cardiac hypertrophy. These findings focus attention on modulating perivascular inflammation and targeting granule cargo release to prevent the development and consequences of LVH.

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To survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing and, whether pharmacological inhibition of such proteins could promote immediate repair of damaged mammalian axons. To this end, I investigated the role of DAG, protein kinase C (PKC) and other DAG-activated proteins in plasmalemmal sealing in B104 neurons in vitro and rat sciatic nerves in vivo. Using dye exclusion to assess Ca2+-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, I now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1, and protein kinase A (PKA). Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes η (nPKCη) and θ (nPKCθ) and, Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or PKA. Sealing probability is further decreased by simultaneously inhibiting nPKCη, nPKCθ and PKA. Extracellular Ca2+, DAG or cAMP analogs do not affect this decrease in sealing. I also report that applying inhibitors of nPKC and PKA to rat sciatic axons crush-severed in vivo under physiological calcium, do not promote immediate repair by polyethylene glycol (PEG), as assessed by compound action potential conduction and dye diffusion through crush sites. These and other data suggest that DAG increases sealing through MARCKS and that nPKCη, nPKCθ and PKA are all required to seal plasmalemmal damage in B104 neurons, and likely all eukaryotic cells. / text