Published papers

AIMS: Cardiovascular diseases are the major cause of mortality in patients with diabetes mellitus. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine and plays an important role in cardiovascular diseases. The objective of this study was to evaluate the relation between the genotypes of the MCP-1 A-2518G polymorphism and the development of carotid atherosclerosis in patients with type 2 diabetes. METHODS: The subjects were 303 unrelated patients who were diagnosed with type 2 diabetes mellitus. To evaluate macroangiopathy, we measured carotid artery intima-media thickness (IMT) by ultrasonography. The MCP-1 A-2518G polymorphism was determined by TaqMan PCR method. RESULTS: IMT in patients with the MCP-1 -2518 AG or GG genotype was significantly greater than the AA-genotype (P=0.007). Simple regression analysis showed that age, systolic blood pressure, LDL-cholesterol, the MCP-1 -2518 AG+GG polymorphism, and HbA1c level were correlated with IMT (P<0.0001, <0.0001, 0.006, 0.007, 0.025, respectively). In multiple regression analysis, the MCP-1 -2518 AG+GG polymorphism was the third strongest independent determinant of IMT in patients with type 2 diabetes (P=0.021), subsequent to age and systolic blood pressure. CONCLUSION: Assessment of the MCP-1 A-2518G polymorphism would be useful in identifying the risk of developing carotid atherosclerosis in patients with type 2 diabetes.

Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

Vascular fibrosis is a major complication of hypertension and atherosclerosis, yet it is largely untreatable. Natriuretic peptides (NPs) repress fibrogenic activation of vascular smooth muscle cells (VSMCs), but the intracellular mechanism mediating this effect remains undetermined. Here we show that inhibition of RhoA through phosphorylation at Ser188, the site targeted by the NP effector cyclic GMP (cGMP)-dependent protein kinase I (cGK I), is critical to fully exert antifibrotic potential. cGK I(+/-) mouse blood vessels exhibited an attenuated P-RhoA level and concurrently increased RhoA/ROCK signaling. Importantly, cGK I insufficiency caused dynamic recruitment of ROCK into the fibrogenic programs, thereby eliciting exaggerated vascular hypertrophy and fibrosis. Transgenic expression of cGK I-unphosphorylatable RhoA(A188) in VSMCs augmented ROCK activity, vascular hypertrophy, and fibrosis more prominently than did that of wild-type RhoA, consistent with the notion that RhoA(A188) escapes the intrinsic inhibition by cGK I. Additionally, VSMCs expressing RhoA(A188) became refractory to the antifibrotic effects of NPs. Our results identify cGK I-mediated Ser188 phosphorylation of RhoA as a converging node for pro- and antifibrotic signals and may explain how diminished cGMP signaling, commonly associated with vascular malfunction, predisposes individuals to vascular fibrosis.

Bariatric surgery has been shown to improve glucose tolerance, although the mechanism has not been fully elucidated. Animal studies have suggested important roles of bile acid (BA) as a regulator of energy homeostasis and glucose metabolism. However, little is known about its role in humans. We investigated the longitudinal changes of BA, incretins, and adipokines after significant weight reduction in 34 Japanese adults with morbid obesity who underwent laparoscopic bariatric surgery. In subjects who underwent malabsorptive or restrictive surgery, body mass index had markedly decreased from 43.0 +/- 6.5 (SD) to 37.8 +/- 5.7 kg/m(2) and from 45.3 +/- 11.2 to 41.5 +/- 10.5 kg/m(2), respectively, at 1 month after surgery. Glycated hemoglobin decreased from 6.1% +/- 1.5% to 5.2% +/- 0.4% and from 6.2% +/- 1.3% to 5.4% +/- 0.7%, and total BA level increased from 3.1 +/- 3.5 to 7.2 +/- 5.3 mumol/L and from 3.2 +/- 2.6 to 9.4 +/- 10.0 mumol/L, respectively. At baseline, serum concentration of primary BA was positively correlated with plasma gastric inhibitory polypeptide level (r = 0.548, P = .001); and change in primary BA level was positively correlated with changes in plasma gastric inhibitory polypeptide (r = 0.626, P = .001) and serum immunoreactive insulin level (r = 0.592, P = .002) at 1 month after surgery. Furthermore, plasma glucagon-like peptide-1 and serum high-molecular weight adiponectin levels increased in both surgeries. These hormonal changes might explain the mechanism(s) of improved glucose tolerance after bariatric surgery in morbidly obese subjects.

Hypertension is a disease which affects over 26.4% of the world adult population, therefore novel approaches to the prevention and treatment of this disease need to be examined. Previous studies from our and other laboratories have shown that treatment of spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats with a renin-angiotensin system (RAS) inhibitor during the 'critical period' in hypertension development results in prevention of the later development of hypertension. In humans, Julius et al. reported similar findings in the landmark TROPHY study. Recently, we reported that 'pulse' treatment of SHR with high-dose angiotensin receptor blocker (ARB) is effective in causing sustained reduction of already established hypertension, even when the treatment was started after the 'critical period'. These results suggest the possibility that 'regression' of established hypertension may become feasible, and we have started a prospective, multicenter clinical study (STAR CAST study) to examine this possibility. In our animal studies, we found that treatment of rats during the 'critical period' with an ARB inhibits the development of renal arteriolar hypertrophy. Moreover, a high-dose angiotensin blocker caused a remarkable reversal of renal arteriolar hypertrophy in SHR, which was associated with changes in microvascular MMP expression. These results suggest that changes in the renal microvasculature may have an important role in the mechanisms of hypertension prevention and regression by ARB.

Patients undergoing hemodialysis are immunocompromised and can suffer from pneumonia with various pathogens in nosocomial conditions. We investigated the fundamental information on the characteristics of hemodialysis inpatients and nosocomial pneumonia. We surveyed 1803 hemodialysis patients admitted to our university hospital between 2001 and 2007. The mean patient age was 64.8 years and the average period of hospitalization was 28.1 days, which was considerably longer than the average stay in our hospital (14.2 days). Patients were admitted to many different departments and for various reasons. We isolated 391 microorganisms from the sputum of 120 pneumonia patients undergoing hemodialysis, including Candida albicans, methicillin-resistant Staphylococcus aureus, and Staphylococcus epidermidis, which were the leading three isolates. From these 120 patients, a total of 199 pathogens were identified as being responsible for the pneumonia. Multi-drug resistant Stenotrophomonas maltophilia was found to be susceptible to a new fluoroquinolone, but is resistant to older generation quinolones. Out of the 120 patients with pneumonia, 12 out of 18 patients infected with S. maltophilia died, indicating the highest fatality rate for this pathogen. In this survey, we found that hemodialysis patients were hospitalized for long periods, and for various reasons in many departments. They suffered from nosocomial pneumonia caused by multi-drug resistant pathogens, including S. maltophilia. For pneumonia due to S. maltophilia, new generation fluoroquinolones can be the treatment of choice, although S. maltophilia-related pneumonia should be treated very carefully because of its high fatality rate.

The binding of prorenin to the (pro)renin receptor triggers 2 major pathways: a nonproteolytic conformational change in prorenin to its active form (angiotensin II-dependent pathway) and an intracellular pathway via the (pro)renin receptor itself (angiotensin II-independent pathway). In diabetic animals, an increased plasma prorenin level not only causes the generation of angiotensin II via the angiotensin II-dependent pathway, it also stimulates the transliteration receptors own intracellular signaling pathway in a manner that is independent of the generated angiotensin II. Thus, the administration of a "handle" region peptide (HRP), which acts as a decoy peptide and competitively inhibits the binding of prorenin to the (pro)renin receptor, has a beneficial effect in the kidneys of diabetic animals with low plasma renin levels. However, the benefits of HRP are slightly reduced in animal models of essential hypertension with relatively high plasma renin levels, and these benefits disappear altogether in animal models of hypertension with extremely high plasma renin levels. Thus, in the kidneys of animal models of diabetes and/or hypertension, both renin and prorenin competitively bind to the (pro)renin receptor and contribute to the pathophysiology of nephropathy. Consequently, renin, prorenin and the (pro) renin receptor may be important therapeutic targets for the prevention and regression of nephropathy in patients with diabetes and/or hypertension.

AIM: Telmisartan, an angiotensin II receptor blocker (ARB), was reported to have partial peroxisome proliferator-activated receptor gamma (PPAR gamma) activity in vitro. Also, adipocyte-derived protein adiponectin, especially its high molecular weight (HMW) form, has been reported to have beneficial effects on insulin resistance and atherosclerosis. We investigated the effects of 3-month telmisartan therapy on various metabolic parameters, including serum HMW adiponectin and high-sensitivity C-reactive protein (hs-CRP) levels in male hypertensive subjects with abdominal obesity. METHODS: This study included 19 Japanese male hypertensive subjects, aged 51.2+/-7.6 (mean+/-SD) years, and body mass index 27.7+/-4.1 kg/m(2). In these subjects, 14 were naive to telmisartan treatment (40.0+/-15.7 mg daily), and 5 were changed from other ARBs to telmisartan. Serum HMW adiponectin concentration was assayed using HMW-selective ELISA kit. RESULTS: In all 19 subjects, systolic/diastolic blood pressure (BP) decreased from 153/98 to 134/85 mmHg (p<0.001 for both). Serum HMW-adiponectin level increased from 2.06+/-0.81 to 2.40+/-0.96 microg/mL (+16.4%, p=0.017). Body weight, glucose, insulin, lipids and hs-CRP did not change during the study period, and there were no adverse effects in any subject. In the newly administered group (n=14), the results were almost the same: BP decreased from 155/98 to 134/84 mmHg (p=0.0015 for both), and serum HMW-adiponectin level increased from 2.07+/-0.68 to 2.39+/-0.99 microg/mL (+15.5%, p=0.089). CONCLUSION: These data suggest that telmisartan therapy is efficient for controlling BP, and may exert beneficial effects on HMW adiponectin in male hypertensive subjects with abdominal obesity.

Most type 1 diabetes mellitus is caused by autoimmune pancreatic beta-cell destruction. Several antigens such as insulin, glutamic acid decarboxylase (GAD) and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) are considered to take part in the autoimmune destructive process. Because the role of GAD in the disease process of type 1 diabetes is still controversial, we investigated the disease phenotype upon in vivo induction of whole GAD65 reactivity using a GAD65 homo knockout NOD splenocytes to NOD-scid transfer system. Splenocytes from 8 to 10-week-old female GAD65 homo knockout (=KOT splenocytes) or age-matched wild type (=WTT splenocytes) NOD mice were transferred into female NOD-scid recipients. As compared to recipients of WTT splenocytes, the onset of diabetes in recipients of KOT splenocytes was significantly delayed (p<0.001). Moreover, TGF-beta expression was enhanced in the pancreas from recipients of KOT splenocytes. Splenocytes from recipients of KOT splenocytes produced IL-10 (/IFN-gamma) upon GAD65 stimulation, whereas those from recipients of WTT splenocytes did not. Based upon these results, we propose that anti-whole GAD65-reactive T cells have the ability to regulate the development of type 1 diabetes.

Glucagon-like peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b(+)Gr-1(low) macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b(+)/Gr-1(-) and CD11b(+)/Gr-1(high) cells, but not CD11b(+)/Gr-1(low) cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic cerulein-administered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b(+)-cell migration to the pancreas is critically involved in chronic pancreatitis-mediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion.

AIM: Clinical studies have suggested that renin-angiotensin inhibitors are effective for the prevention of atherosclerosis progression, but the results for the regression of established lesions are equivocal. The aim of this study was to examine the effects of different doses of the angiotensin receptor blocker (ARB) candesartan on the regression of atherosclerosis and lipid-induced nephropathy in apolipoprotein E (apoE)-deficient spontaneously hyperlipidemic (SHL) mice. METHODS AND RESULTS: Male SHL were given an atherogenic diet together with salt loading to induce atherosclerosis. The mice were then treated with various doses of candesartan (0-50 mg/kg/d) for 12 weeks. Treatment with high-dose candesartan caused clear regression of atherosclerotic plaques in the aorta, which was not observed with normal-dose candesartan. Biglycan and ACAT1 expression were significantly decreased, and aortic free cholesterol: cholesterol ester ratios were increased in these mice. Treatment of cultured THP-1 macrophages in vitro with candesartan resulted in a similar decrease in ACAT1 expression. In the kidney, glomerular lipid accumulation, mesangial expansion, and albuminuria were significantly regressed after treatment with high-dose candesartan, while biglycan and ACAT1 expressions were decreased. CONCLUSION: These results suggest that regression of established atherosclerosis lesions in ApoE-deficient mice is feasible using high-dose candesartan, by mechanisms involving (i) a decrease in the lipid-retaining proteoglycan biglycan, and (ii) suppression of ACAT1 expression resulting in increased free cholesterol for lipid release.

There has been concern as to whether dipeptidyl peptidase-4 (DPP-4) inhibitors can be used safely in patients with relatively good glycemic control. This study, approved by the institutional review board of Hanzoumon Diabetes City Atlas Clinic, examined whether DPP-4 inhibitor sitagliptin could safely achieve good glycemic control without severe hypoglycemia by employing the "added food" concept. The subjects were 60 patients (46 men and 14 women) with type 2 diabetes who started sitagliptin therapy during a 1-month period from December 15, 2009 to January 15, 2010. They were recommended to have added food between meals to prevent hypoglycemia, while maintaining the same daily calorie intake. HbA(1c) decreased from 7.1 +/- 1.2% to 6.5 +/- 0.6% after 6 months of sitagliptin treatment (p < 0.001). In patients with a baseline HbA(1c) <7%, it decreased from 6.5 +/- 0.3% to 6.1 +/- 0.4% (p < 0.001). Systolic blood pressure was significantly reduced from 127.7 +/- 17.0 to 122.7 +/- 17.9 mmHg in the patients with a baseline HbA(1c) < 7% (p = 0.018). However, body weight increased by approximately 900 g and high-density lipoprotein cholesterol decreased significantly from 1.57 +/- 0.46 to 1.43 +/- 0.35 mmol/L (p < 0.01) in the patients concomitantly receiving sulfonylureas with sitagliptin. Excellent glycemic control was achieved by sitagliptin treatment together with the added food concept. However, combined use of sitagliptin with sulfonylureas requires attention to weight gain and the lipid profile. Further clinical studies will elucidate whether sitagliptin can decrease cardiovascular events as well as normalizing blood glucose and lowering the blood pressure.

Introduction. There is no report about risk factors for renal deterioration according to the clinical stage, divided by the estimated glomerular filtration rate (eGFR) in type 2 diabetes. Materials and Methods. We evaluated the factors correlated with the annual eGFR decline in 1303 subjects with type 2 diabetes whose eGFR was >/=30 mL/min/1.73 m(2). eGFR strata were defined by baseline eGFR value as follows: stratum 1: >/=90, stratum 2: >/=60, <90, and stratum 3: >/=30, <60. Results. The annual eGFR decline was 2.3 +/- 5.4 mL/min/1.73 m(2) in overall subjects. Multiple linear regression analysis demonstrated that age, male sex, systolic blood pressure, logarithmically transformed albumin excretion rate (AER), eGFR strata, and hemoglobin concentration were significantly correlated with the annual eGFR decline. When stratified by eGFR, the factors that showed a significant correlation were different among eGFR strata. AER was significantly correlated with annual eGFR decline in all eGFR strata. Hemoglobin concentration showed a significant correlation only in the advanced eGFR stratum. Conclusion. The factors correlated with the annual eGFR decline were different among eGFR strata in type 2 diabetes mellitus, and hemoglobin concentration and AER were important factors for renal deterioration, especially in the advanced eGFR stratum.

It has been reported that beta cell function progressively declines in patients with type 2 diabetes. The objective of this study was to assess the effect of obesity on declining beta cell function after diagnosis of type 2 diabetes. We conducted a cross-sectional study of 689 consecutive subjects with type 2 diabetes who were admitted to our hospital from 2000 to 2007. Fasting and postprandial serum C-peptide immunoreactivity (CPR) and urinary CPR levels had been measured during admission. The subjects were stratified according to BMI and time since diagnosis. CPR index was calculated as CPR (ng/mL) / plasma glucose (mg/dL) x 100. All CPR measurements were significantly higher in the 263 obese (BMI >/=25) subjects compared to the 426 lean subjects (BMI <25). There was a significant negative correlation between CPR indices and duration of diabetes, suggesting a progressive decline in beta cell function after diagnosis of type 2 diabetes. However, this decline was more apparent in obese subjects (postprandial CPR index 0.059/year) compared to lean subjects (0.025/year). The significant difference in serum CPR indices between the lean and obese subjects was lost in subjects more than 10 years after diagnosis. In conclusion, our observations suggest that beta cell function shows a greater progressive decline in obese subjects than in lean subjects with type 2 diabetes. Treatment of obesity may be an important strategy to preserve beta cell function in patients with type 2 diabetes.

BACKGROUND: Besides well-established roles of bile acids (BA) in dietary lipid absorption and cholesterol homeostasis, it has recently become clear that BA is also a biological signaling molecule. We have shown that strategies aimed at activating TGR5 by increasing the BA pool size with BA administration may constitute a significant therapeutic advance to combat the metabolic syndrome and suggest that such strategies are worth testing in a clinical setting. Bile acid binding resin (BABR) is known not only to reduce serum cholesterol levels but also to improve glucose tolerance and insulin resistance in animal models and humans. However, the mechanisms by which BABR affects glucose homeostasis have not been established. We investigated how BABR affects glycemic control in diet-induced obesity models. METHODS AND FINDINGS: We evaluated the metabolic effect of BABR by administrating colestimide to animal models for the metabolic syndrome. Administration of BABR increased energy expenditure, translating into significant weight reduction and insulin sensitization. The metabolic effects of BABR coincide with activation of cholesterol and BA synthesis in liver and thermogenesis in brown adipose tissue. Interestingly, these effects of BABR occur despite normal food intake and triglyceride absorption. Administration of BABR and BA had similar effects on BA composition and thermogenesis, suggesting that they both are mediated via TGR5 activation. CONCLUSION: Our data hence suggest that BABR could be useful for the management of the impaired glucose tolerance of the metabolic syndrome, since they not only lower cholesterol levels, but also reduce obesity and improve insulin resistance.

Coronary artery aneurysms are rare complications of autosomal dominant polycystic kidney disease (ADPKD), and their pathogenesis remains poorly understood. We report an autopsy case of a 64-year-old ADPKD patient with an asymptomatic, large (4 cm in diameter) saccular aneurysm arising from the left circumflex (LCX) branch of the coronary artery with only mild atherosclerotic changes. Autopsy also revealed small, focal defects of media with or without microaneurysm formation in the LCX, mesenteric and renal arteries, and a fibromuscular dysplasia-like lesion with microaneurysm in the common iliac artery. Since polycystin-1 and -2 are expressed in arterial smooth-muscle cells, these findings imply that abnormal polycystin expression in ADPKD initially causes the focal medial defects, some of which might later progress to microaneurysms and then overt aneurysms. To the best of our knowledge, this is the first description of the pathologic findings of an ADPKD-associated coronary aneurysm and its probable precursor lesions in arteries.

The renin-angiotensin-aldosterone system(RAAS) plays a crucial role in the regulation of physiological homeostasis and diseases such as hypertension, coronary artery disease and chronic renal failure. In this cascade, the ACE/Ang II/AT1 receptor axis induces pathological effects, such as vasoconstriction, cell proliferation and fibrosis. Recently the ACE2/Ang(1-7)/Mas receptor axis has been recognized as a negative regulator of the RAAS. ACE2 metabolizes Ang II into Ang(1-7), which has opposite properties of Ang II through Mas receptor activation. Both animal and human studies provide strong evidence that the ACE2/Ang(1-7)/Mas receptor axis is protective for end-organ damage. Therefore, the ACE2/Ang(1-7)/Mas receptor axis could be a therapeutic target for coronary artery disease and chronic renal failure.

The renin-angiotensin system (RAS) plays a central role in the control of systemic blood pressure. Since the 1950s, both laboratory and clinical studies have been performed to test the efficacy of vaccines against the RAS for the management of hypertension. Although initial studies using renin vaccines and angiotensin I vaccines were unsuccessful, a recent phase IIa study has shown that vaccination against angiotensin II is effective for the treatment of patients with essential hypertension. Results from animal studies have also suggested that vaccination against the AT1 receptor may be effective not only for lowering blood pressure, but also for the prevention of hypertensive end-organ damage. In the future, the RAS may be an important target for the development of vaccines not only for the treatment, but also for the prevention of hypertension and hypertensive complications.

Chronic inflammation in adipose tissue contributes to obesity-related insulin resistance. The 3-phosphoinositide-dependent protein kinase 1 (Pdk1)/forkhead transcription factor (Foxo1) pathway is important in regulating glucose and energy homeostasis, but little is known about this pathway in adipose tissue macrophages (ATMs). To investigate this, we generated transgenic mice that carried macrophage/granulocyte-specific mutations, including a Pdk1 knockout (LysMPdk1(-/-)), a Pdk1 knockout with transactivation-defective Foxo1 (Delta256LysMPdk1(-/-)), a constitutively active nuclear (CN) Foxo1 (CNFoxo1(LysM)), or a transactivation-defective Foxo1 (Delta256Foxo1(LysM)). We analyzed glucose metabolism and gene expression in ATM populations isolated with fluorescence-activated cell sorting. The LysMPdk1(-/-) mice exhibited elevated M1 macrophages in adipose tissue and insulin resistance. Overexpression of transactivation-defective Foxo1 rescued these phenotypes. CNFoxo1(LysM) promoted transcription of the C-C motif chemokine receptor 2 (Ccr2) in ATMs and increased M1 macrophages in adipose tissue. On a high-fat diet, CNFoxo1(LysM) mice exhibited insulin resistance. Pdk1 deletion or Foxo1 activation in bone marrow-derived macrophages abolished insulin and interleukin-4 induction of genes involved in alternative macrophage activation. Thus, Pdk1 regulated macrophage infiltration by inhibiting Foxo1-induced Ccr2 expression. This shows that the macrophage Pdk1/Foxo1 pathway is important in regulating insulin sensitivity in vivo.