Mariko Kyutoku

Role of periostin in cancer progression and metastasis: Inhibition of breast cancer progression and metastasis by anti-periostin antibody in a murine model

Periostin (PN), a secreted adhesion-related protein expressed in the periosteum and periodontal ligaments, acts as a critical regulator of the formation and maintenance of bone and teeth, and also plays an important role in tumorigenesis. Although PN is highly expressed in various types of human cancers, its function is still unclear. In this study, we focused on the exon 17 region of PN, which is alternatively spliced out. To investigate the function of full-length PN with exon 17, we produced a neutralizing antibody (PN1-Ab) against the peptide encoded by exon 17. In vivo, administration of PN1-Ab significantly inhibited the growth of primary tumors as well as metastatic tumors, associated with prevention of bone destruction, resulting in increased survival of mice. Consistent with in vivo data, the present in vitro study demonstrated that addition of full-length PN significantly inhibited cell adhesion and detached adherent cells, while PN1-Ab inhibited the action of PN in a dose-dependent manner. In addition, PN1-Ab significantly inhibited the proliferation, migration and invasion of 4T1 mouse breast cancer cells, which produced PN. Interestingly, PN1-Ab also inhibited the differentiation of osteoclasts. Overall, the present study demonstrated that PN plays a pivotal role in the progression and metastasis of breast cancer. Since administration of PN1-Ab prolonged cell survival through inhibition of the progression and metastasis of 4T1 cells, further development of the PN1-Ab such as generation of a humanized antibody may provide a new therapeutic agent against breast cancer.

Decrease in blood pressure and regression of cardiovascular complications by angiotensin II vaccine in mice.

Vaccines have been recently developed to treat various diseases such as cancer, rheumatoid arthritis and Alzheimer’s disease in addition to infectious diseases. However, before use in the clinical setting, vaccines targeting self-antigens must be demonstrated to be effective and safe, evoking an adequate humoral immune response from B cells while avoiding T cell activation in response to self. Although the vaccine targeting angiotensin II (Ang II) is efficient in rodents and humans, little is known regarding the immunological activation and safety of the vaccine. In this study, we evaluated the efficiency and safety of an Ang II peptide vaccine in mice. Immunization with Ang II conjugated to keyhole limpet hemocyanin (KLH) successfully induced the production of anti-Ang II antibody, which blocked Ang II signaling in human aortic smooth muscle cells. However, Ang II itself did not activate T cells, as assessed by the proliferation and lymphokine production of T cells in immunized mice, whereas KLH activated T cells. In an Ang II-infused model, the non-immunized mice showed high blood pressure (BP), whereas the immunized mice (Ang II-KLH) showed a significant decrease in systolic BP, accompanied by significant reductions in cardiac hypertrophy and fibrosis. Importantly, anti-Ang II antibody titer was not elevated even after the administration of large amounts of Ang II, indicating that Ang II itself boosted antibody production, most likely due to less activation of T cells. In addition, no accumulation of inflammatory cells was observed in immunized mice, because endogenous Ang II would not activate T cells after immunization with Ang II-KLH. Taken together, these data indicate that vaccines targeting Ang II might be effective to decrease high BP and prevent cardiovascular complications without severe side effects.

Role of Central Nervous System Periostin in Cerebral Ischemia

Background and Purpose— Although periostin, an extracellular matrix glycoprotein, plays pivotal roles in survival, migration, and regeneration in various cells, its expression and function in the brain are still unknown. Here, we investigated the expression and role of periostin in the ischemic brain. Methods— Expression of full-length periostin (periostin 1 [Pn1]) and its splicing variant lacking exon 17 (periostin 2 [Pn2]) was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical staining in male C57BL6/J mice. The actions of periostin were examined in adult primary neuronal culture and in a transient middle cerebral artery occlusion (tMCAo) model. Results— Expression of Pn2, but not of Pn1, mRNA was markedly changed after tMCAo. Pn2 mRNA was decreased in the ischemic core at 3 hours after ischemia. At 24 hours, Pn2 mRNA was significantly increased in both the peri-ischemic and ischemic regions. Periostin was mainly observed in neurons in normal brain. However, neuronal expression of periostin was decreased temporarily in the ischemic region, but increased in astrocytes and around endothelial cells at 24 hours after tMCAo. Of importance, intracerebroventricular injection of Pn2 resulted in a significant reduction in infarct volume at 24 hours after tMCAo associated with phosphorylation of Akt. Also, the Pn2-treated mice survived longer until 1 week after tMCAo. Pn2 significantly inhibited neuronal death under hypoxia and stimulated neurite outgrowth. Conclusions— Here, we demonstrated that periostin was expressed in the brain, and exogenous Pn2 exhibited neuroprotective effects and accelerated neurite outgrowth. Additional studies on periostin may provide new insights into the treatment of ischemic stroke.

Development of novel DNA vaccine for VEGF in murine cancer model

We developed DNA vaccine for vascular endothelial growth factor (VEGF), which may provide the therapeutic option instead of anti-VEGF antibody, bevacizumab. Plasmid containing VEGF mini-gene was constructed in the insertion of B-cell epitope of Hepatitis B core protein [HBc-VEGF], which was an epitope carrier. High titer of anti-VEGF antibody was observed in BALB/c mice which were intramuscularly immunized with HBc-VEGF by electropolator. In mice inoculated with colon 26 cells, tumor volume and microvessel density was decreased in HBc-VEGF with a significant prolonged survival. Co-treatment of purified IgG from immunized mice with HBc-VEGF showed in vitro neutralizing activity for VEGF-induced ERK phosphorylation and tube formation in cultured endothelial cells. Furthermore, intravitreally injection of this purified IgG reduced the neovessel formation in the mouse oxygen-induced retinopathy and laser-induced choroidal neovascularization models. These results first provided that DNA vaccine against VEGF possessed the anti-angiogenic effect, leading to prolonged survival in mouse cancer model.

Long-Term Reduction of High Blood Pressure by Angiotensin II DNA Vaccine in Spontaneously Hypertensive Rats

Recent research on vaccination has extended its scope from infectious diseases to chronic diseases, including Alzheimer disease, dyslipidemia, and hypertension. The aim of this study was to design DNA vaccines for high blood pressure and eventually develop human vaccine therapy to treat hypertension. Plasmid vector encoding hepatitis B core-angiotensin II (Ang II) fusion protein was injected into spontaneously hypertensive rats using needleless injection system. Anti-Ang II antibody was successfully produced in hepatitis B core-Ang II group, and antibody response against Ang II was sustained for at least 6 months. Systolic blood pressure was consistently lower in hepatitis B core-Ang II group after immunization, whereas blood pressure reduction was continued for at least 6 months. Perivascular fibrosis in heart tissue was also significantly decreased in hepatitis B core-Ang II group. Survival rate was significantly improved in hepatitis B core-Ang II group. This study demonstrated that Ang II DNA vaccine to spontaneously hypertensive rats significantly lowered high blood pressure for at least 6 months. In addition, Ang II DNA vaccines induced an adequate humoral immune response while avoiding the activation of self-reactive T cells, assessed by ELISPOT assay. Future development of DNA vaccine to treat hypertension may provide a new therapeutic option to treat hypertension.

Cilnidipine, but not amlodipine, ameliorates osteoporosis in ovariectomized hypertensive rats through inhibition of the N-type calcium channel.

Both osteoporosis and high blood pressure are major diseases in aging populations. Recent studies demonstrated that some antihypertensive drugs reduced the risk of bone fracture in elderly patients. Although calcium channel blockers (CCB) are widely used as first-line antihypertensive agents, there is no evidence that they prevent osteoporosis. In this study, we investigated the effects of two types of CCB on bone metabolism: cilnidipine (L-/N-type CCB), which suppresses norepinephrine release from the sympathetic nerve, and amlodipine (L-type CCB). In ovariectomized female spontaneous hypertensive rats, administration of cilnidipine, but not amlodipine, resulted in a significant increase in the ratio of alkaline phosphatase to tartrate-resistant acid phosphatase (TRAP) and a decrease in the number of osteoclasts, as assessed by TRAP staining in the proximal tibia. Bone mineral density, moreover, was significantly higher in the cilnidipine group as compared with the amlodipine group and was associated with a significant decrease in a urinary collagen degradation product (deoxypyridinoline). The degree of prevention of osteoporosis by cilnidipine was similar to that of carvedilol (a β-blocker) because β-blockers reduce fracture risks though the inhibition of osteoclast activation. Interestingly, these effects cannot be attributed to the reduction of blood pressure because all three drugs significantly decreased blood pressure. In contrast, both cilnidipine and carvedilol, but not amlodipine, significantly decreased heart rate, indicating that both cilnidipine and carvedilol suppressed sympathetic nervous activity. Overall, our present data showed that cilnidipine (L-/N-type CCB) ameliorated osteoporosis in ovariectomized hypertensive rats. These pleiotropic effects of antihypertensive drugs such as cilnidipine and carvedilol might provide additional benefits in the treatment of hypertensive postmenopausal women.

Inhibition of Neointima Formation through DNA Vaccination for Apolipoprotein(a): A New Therapeutic Strategy for Lipoprotein(a)

Lipoprotein(a) [Lp(a)] is an unique lipoprotein consisting of the glycoprotein apolipoprotein(a) [apo(a)] in low-density lipoprotein. Although Lp(a) is a well-known independent risk factor for cardiovascular disease; however, there is no drugs to decrease plasma Lp(a) level. Thus, to inhibit the biological activity of Lp(a), we developed DNA vaccine for apo(a) by the targeting to the selected 12 hydrophilic amino acids in the kringle-4 type 2 domain of apo(a). Hepatitis B virus core protein was used as an epitope carrier to enhance the immunogenicity. Intramuscular immunization with apo(a) vaccine resulted in the significant inhibition of neointima formation in carotid artery ligation model using Lp(a) transgenic mice, associated with anti-apo(a) antibody and decrease in vascular Lp(a) deposition. Overall, this study provided the first evidence that the pro-atherosclerotic actions of Lp(a) could be prevented by DNA vaccine directed against apo(a), suggesting a novel therapeutic strategy to treat cardiovascular diseases related to high Lp(a).

Nifedipine prevents hepatic fibrosis in a non-alcoholic steatohepatitis model induced by an L-methionine-and choline-deficient diet

Recent reports have shown that nifedipine, a calcium channel blocker, increases peroxisome proliferator-activated receptor-γ (PPARγ) activity. Since PPARγ agonists, such as pioglitazone and rosiglitazone, are effective in reducing non-alcoholic steatohepatitis (NASH) and cirrhosis in animal models, we examined the protective effects of nifedipine, as compared with bezafibrate, a PPARα agonist, in a NASH model induced by an L-methionine- and choline-deficient (MCD) diet. An MCD diet for 20 weeks changed the color of the rat liver to yellow with an irregular surface, whereas the color of the liver in both the bezafibrate and nifedipine treatment groups was markedly changed to yellow-brown with a smooth surface. Furthermore, nifedipine, as well as bezafibrate, significantly prevented liver fibrosis induced by an MCD diet, as assessed by Massons trichrome staining, accompanied by a significant decrease in serum AST. Overall, nifedipine treatment resulted in an improvement in NASH, similar to bezafibrate, in a rat model. In hypertensive patients with metabolic syndrome, nifedipine may provide additional benefits, beyond its blood pressure-lowering effects, to prevent NASH and fatty liver disease.

Links Between Hypertension and Osteoporosis: Benidipine Ameliorates Osteoporosis in Ovariectomized Hypertensive Rats Through Promotion of Osteoblast Proliferation and Inhibition of Osteoclast Differentiation

Recent studies have demonstrated that some antihypertensive drugs reduced the risk of bone fracture. Although calcium channel blockers (CCB) are used as first-line agents, there is no evidence that they prevent osteoporosis. In this study, we investigated the effects of 2 types of CCB, benidipine (L-/T-type CCB) and amlodipine (L-type CCB), on bone metabolism. In ovariectomized SHR, administration of benidipine resulted in a significant increase in the ratio of ALP to TRAP and a decrease in the number of osteoclasts in the tibia. Moreover, bone mineral density was significantly higher in the benidipine group compared with the amlodipine group, associated with a significant decrease in urinary deoxypyridinoline. In an in vitro study, benidipine promoted ALP expression and decreased receptor activator of NF-kB (RANK) ligand expression of human osteoblasts, indicating suppression of osteoclast differentiation. These pleiotropic effects of antihypertensive drugs such as benidipine might provide additional benefits in treating hypertensive postmenopausal women.

819 DEVELOPMENT OF ANGIOTENSIN II DNA VACCINE FOR HYPERTENSION

Objectives: The aim of this study is to design DNA vaccines for high blood pressure (BP) toward to human vaccine therapy to treat hypertension. Design and methods: We selected angiotensin II (Ang II) as a target antigen. Plasmid vector encoding Hepatitis B core (HBc)-Ang II fusion protein was constructed, as HBc forms sphere-like structure by self-aggregation and the presented fusion peptide in the position of B cell epitope of HBc protein could induce high titer antibody to the target peptide. Plasmid DNA (HBc-Ang II or HBc) or saline were injected to the back skin of spontaneously hypertensive rats (SHR) three times (at 0, 2, 4 weeks) by needle less injection system. Results: Anti-Ang II antibody was successfully produced, and sustained at least up to 6 months. Consistently, systolic BP was lower in HBc-Ang II group after the immunization (at 12 weeks: 188.1 ± 15.8 mmHg in HBc-Ang II group vs. 221.5 ± 14.6 mmHg in HBc group, P < 0.01), and BP reduction was continued at least up to 6 months. Of importance, perivascular fibrosis in heart tissue was significantly reduced in HBc-Ang II group. T cell activity of cytokine production responded to Ang II or angiotensinogen was not detected without any apparent pathologically toxic observation. Conclusions: The present study demonstrated that Ang II DNA vaccine to SHR significantly lowered high BP at least up to 6 months. Future development of DNA vaccine to hypertension might provide new therapeutic option to treat hypertensive population.