Tsutomu Nakahara

Rapid vascular regrowth in tumors after reversal of VEGF inhibition

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Effect of Inhibition of Vascular Endothelial Growth Factor Signaling on Distribution of Extravasated Antibodies in Tumors

Antibodies and other macromolecular therapeutics can gain access to tumor cells via leaky tumor vessels. Inhibition of vascular endothelial growth factor (VEGF) signaling can reduce the vascularity of tumors and leakiness of surviving vessels, but little is known about how these changes affect the distribution of antibodies within tumors. We addressed this issue by examining the distribution of extravasated antibodies in islet cell tumors of RIP-Tag2 transgenic mice and implanted Lewis lung carcinomas using fluorescence and confocal microscopic imaging. Extravasated nonspecific immunoglobulin G (IgG) and antibodies to fibrin or E-cadherin accumulated in irregular patchy regions of stroma. Fibrin also accumulated in these regions. Anti-E-cadherin antibody, which targets epitopes on tumor cells of RIP-Tag2 adenomas, was the only antibody to achieve detectable levels within tumor cell clusters at 6 hours after i.v. injection. Treatment for 7 days with AG-013736, a potent inhibitor of VEGF signaling, reduced the tumor vascularity by 86%. The overall area density of extravasated IgG/antibodies decreased after treatment but the change was less than the reduction in vascularity and actually increased when expressed per surviving tumor vessel. Accumulation of anti-E-cadherin antibody in tumor cell clusters was similarly affected. The patchy pattern of antibodies in stroma after treatment qualitatively resembled untreated tumors and surprisingly coincided with sleeves of basement membrane left behind after pruning of tumor vessels. Together, the findings suggest that antibody transport increases from surviving tumor vessels after normalization by inhibition of VEGF signaling. Basement membrane sleeves may facilitate this transport. Antibodies preferentially distribute to tumor stroma but also accumulate on tumor cells if binding sites are accessible.

Dynamic nonlinear vago-sympathetic interaction in regulating heart rate

SummaryAlthough the characteristics of the static interactions between the sympathetic and parasympathetic nervous systems in regulating heart rate have been well established, how the dynamic interaction modulates the heart rate response remains unknown. Thus, we investigated the dynamic interaction by estimating the transfer function from nerve stimulation to heart rate, using band-limited Gaussian white noise, in anesthetized rabbits. Concomitant tonic vagal stimulation at 5 and 10Hz increased the gain of the transfer function relating dynamic sympathetic stimulation to heart rate by 55.0% ± 40.1% and 80.7% ± 50.5%, respectively (P < 0.05). Concomitant tonic sympathetic stimulation at 5 and 10Hz increased the gain of the transfer function relating dynamic vagal stimulation to heart rate by 18.2% ± 17.9% and 24.1% ± 18.0%, respectively (P < 0.05). Such bidirectional augmentation was also observed during simultaneous dynamic stimulation of the sympathetic and vagal nerves independent of their stimulation patterns. Because of these characteristics, changes in sympathetic or vagal tone alone can alter the dynamic heart rate response to stimulation of the other nerve. We explained this phenomenon by assuming a sigmoidal static relationship between autonomic nerve activity and heart rate. To confirm this assumption, we identified the static and dynamic characteristics of heart rate regulation by a neural network analysis, using large-amplitude Gaussian white noise input. To examine the mechanism involved in the bidirectional augmentation, we increased cytosolic adenosine 3′,5′-cyclic monophosphate (cAMP) at the postjunctional effector site by applying pharmacological interventions. The cAMP accumulation increased the gain of the transfer function relating dynamic vagal stimulation to heart rate. Thus, accumulation of cAMP contributes, at least in part, to the sympathetic augmentation of the dynamic vagal control of heart rate.

Increased Brain Angiotensin Receptor in Rats With Chronic High-Output Heart Failure

BACKGROUND The renin-angiotensin system (RAS) plays a key role in the pathophysiology of chronic heart failure (CHF). In rats, we reported that CHF enhances dipsogenic responses to centrally administered angiotensin I, and central inhibition of the angiotensin-converting enzyme (ACE) prevents cardiac hypertrophy in CHF. This suggests that the brain RAS is activated in CHF. To clarify the mechanism of the central RAS activation in CHF, we examined brain ACE and the angiotensin receptor (AT) among rats with CHF. METHODS AND RESULTS We created high-output heart failure in 22 male Sprague-Dawley rats by aortocaval shunt. Four weeks after surgery, we examined ACE mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and AT by binding autoradiography. ACE mRNA levels were not significantly increased in the subfornical organ (SFO), the hypothalamus, or in the lower brainstem of CHF rats (n = 5) compared with sham-operated rats (SHM) (n = 6). Binding densities for type 1 AT (AT1) in the SFO (P < .05), paraventricular hypothalamic nuclei (P < .05), and solitary tract nuclei (P < .05) were higher in rats with CHF (n = 5) than in SHM rats (n = 6). Thus, in rats with CHF, AT1 expression is increased in brain regions that are closely related to water intake, vasopressin release, and hemodynamic regulation. CONCLUSIONS The fact that AT1 expression was upregulated in important brain regions related to body fluid control in CHF rats indicates that the brain is a major site of RAS action in CHF rats and, therefore, a possible target site of ACE-inhibitors in the treatment of CHF.

Histological protection by cilnidipine, a dual L/N-type Ca2+ channel blocker, against neurotoxicity induced by ischemia–reperfusion in rat retina

Although a blockade or lack of N-type Ca(2+) channels has been reported to suppress neuronal injury induced by ischemia-reperfusion in several animal models, information is still limited regarding the neuroprotective effects of a dual L/N-type Ca(2+) channel blocker, cilnidipine. We histologically examined the effects of cilnidipine on neuronal injury induced by ischemia-reperfusion, intravitreous N-methyl-D-aspartate (NMDA) (200nmol/eye) and intravitreous NOC12 (400nmol/eye), an nitric oxide donor, in the rat retina, and compared its effects with those of omega-conotoxin MV IIA, an N-type Ca(2+) channel blocker and amlodipine, an L-type Ca(2+) channel blocker. Morphometric evaluation at 7 days after ischemia-reperfusion showed that treatment with cilnidipine (100microg/kg, i.v. or 0.5pmol/eye, intravitreous injection) prior to ischemia dramatically reduced the retinal damage. Treatment with omega-conotoxin MV IIA before ischemia (0.1pmol/eye, intravitreous injection) significantly reduced the retinal damage. However, amlodipine (30-100microg/kg, i.v. or 0.1-1pmol/eye, intravitreous injection) did not show any protective effects. Treatment with cilnidipine (100microg/kg, i.v.) reduced the retinal damage induced by intravitreous NMDA, but not NOC12. These results suggest that cilnidipine reduces Ca(2+) influx via N-type Ca(2+) channels after NMDA receptors activation and then protects neurons against ischemia-reperfusion injury in the rat retina in vivo. Cilnidipine may be useful as a therapeutic drug against retinal diseases which cause neuronal cell death, such as glaucoma and central retinal vessel occlusion.

Histological protection against ischemia–reperfusion injury by early ischemic preconditioning in rat retina

Brief ischemia was reported to protect various cells against injury induced by subsequent ischemia-reperfusion, and this phenomenon is known as ischemic preconditioning. The aims of the present study were to clarify whether early ischemic preconditioning could be observed in the rat retina by histological examination. Male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 5-60 min before 60 min of ischemia. Additional groups of rats received 10 mg/kg 8-phenyltheophiline and 4.5 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A1 receptor antagonists, 5 mg/kg 5-hydroxydecanoate and 1 mg/kg glibenclamide, ATP-sensitive K+ channel blockers, or 2.5 mg/kg chelerythrine and 0.1 mg/kg bisindolylmaleimide I, protein kinase C inhibitors, 15 or 30 min before preconditioning. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Five minutes of preconditioning ischemia 20-40 min before 60 min of sustained ischemia completely prevented the retinal tissue damage induced by the sustained ischemia. Treatment with 8-phenyltheophylline, DPCPX, 5-hydroxydecanoate, glibenclamide, chelerythrine and bisindolylmaleimide I almost completely reduced the protective effect of early ischemic preconditioning. The results in the present study indicated that early ischemic preconditioning was demonstrated in the rat retina. Stimulation of adenosine receptors, opening of ATP-sensitive K+ channels and activation of protein kinase C might be involved in the underlying protective mechanisms.

Cholinesterase affects dynamic transduction properties from vagal stimulation to heart rate

Recent investigations in our laboratory using a Gaussian white noise technique showed that the transfer function representing the dynamic properties of transduction from vagus nerve activity to heart rate had characteristics of a first-order low-pass filter. However, the physiological determinants of those characteristics remain to be elucidated. In this study, we stimulated the vagus nerve according to a Gaussian white noise pattern to estimate the transfer function from vagal stimulation to the heart rate response in anesthetized rabbits and examined how changes in acetylcholine kinetics affected the transfer function. We found that although increases in the mean frequency of vagal stimulation from 5 to 10 Hz did not change the characteristics of the transfer function, administration of neostigmine (30 microg . kg-1 . h-1 iv), a cholinesterase inhibitor, increased the dynamic gain from 8.19 +/- 3.66 to 11.7 +/- 4.88 beats . min-1 . Hz-1 (P < 0.05), decreased the corner frequency from 0.12 +/- 0.05 to 0.04 +/- 0.01 Hz (P < 0.01), and increased the lag time from 0.17 +/- 0.12 to 0.27 +/- 0.08 s (P < 0.05). These results suggest that the rate of acetylcholine degradation at the neuroeffector junction, rather than the amount of available acetylcholine, plays a key role in determining the dynamic properties of transduction from vagus nerve activity to heart rate.Recent investigations in our laboratory using a Gaussian white noise technique showed that the transfer function representing the dynamic properties of transduction from vagus nerve activity to heart rate had characteristics of a first-order low-pass filter. However, the physiological determinants of those characteristics remain to be elucidated. In this study, we stimulated the vagus nerve according to a Gaussian white noise pattern to estimate the transfer function from vagal stimulation to the heart rate response in anesthetized rabbits and examined how changes in acetylcholine kinetics affected the transfer function. We found that although increases in the mean frequency of vagal stimulation from 5 to 10 Hz did not change the characteristics of the transfer function, administration of neostigmine (30 μg ⋅ kg-1 ⋅ h-1iv), a cholinesterase inhibitor, increased the dynamic gain from 8.19 ± 3.66 to 11.7 ± 4.88 beats ⋅ min-1 ⋅ Hz-1( P < 0.05), decreased the corner frequency from 0.12 ± 0.05 to 0.04 ± 0.01 Hz ( P < 0.01), and increased the lag time from 0.17 ± 0.12 to 0.27 ± 0.08 s ( P < 0.05). These results suggest that the rate of acetylcholine degradation at the neuroeffector junction, rather than the amount of available acetylcholine, plays a key role in determining the dynamic properties of transduction from vagus nerve activity to heart rate.

Simultaneous identification of static and dynamic vagosympathetic interactions in regulating heart rate

We earlier reported that stimulation of either one of the sympathetic and vagal nerves augments the dynamic heart rate (HR) response to concurrent stimulation of its counterpart. We explained this phenomenon by assuming a sigmoidal static relationship between nerve activity and HR. To confirm this assumption, we stimulated the sympathetic and/or vagal nerve in anesthetized rabbits using large-amplitude Gaussian white noise and determined the static and dynamic characteristics of HR regulation by a neural network analysis. The static characteristics approximated a sigmoidal relationship between the linearly predicted and the measured HRs (response range: 212.4 ± 46.3 beats/min, minimum HR: 96.0 ± 28.4 beats/min, midpoint of operation: 196.7 ± 31.3 beats/min, maximum slope: 1.65 ± 0.51). The maximum step responses determined from the dynamic characteristics were 7.9 ± 2.9 and -14.0 ± 4.9 beats ⋅ min-1 ⋅ Hz-1for the sympathetic and the vagal system, respectively. Because of these characteristics, changes in sympathetic or vagal tone alone can alter the dynamic HR response to stimulation of the other nerve.We earlier reported that stimulation of either one of the sympathetic and vagal nerves augments the dynamic heart rate (HR) response to concurrent stimulation of its counterpart. We explained this phenomenon by assuming a sigmoidal static relationship between nerve activity and HR. To confirm this assumption, we stimulated the sympathetic and/or vagal nerve in anesthetized rabbits using large-amplitude Gaussian white noise and determined the static and dynamic characteristics of HR regulation by a neural network analysis. The static characteristics approximated a sigmoidal relationship between the linearly predicted and the measured HRs (response range: 212.4 +/- 46.3 beats/min, minimum HR: 96.0 +/- 28.4 beats/min, midpoint of operation: 196.7 +/- 31.3 beats/min, maximum slope: 1.65 +/- 0.51). The maximum step responses determined from the dynamic characteristics were 7.9 +/- 2.9 and -14.0 +/- 4.9 beats. min-1. Hz-1 for the sympathetic and the vagal system, respectively. Because of these characteristics, changes in sympathetic or vagal tone alone can alter the dynamic HR response to stimulation of the other nerve.

Protective effect of all-trans retinoic acid on NMDA-induced neuronal cell death in rat retina.

We histologically examined the effects of all-trans retinoic acid (ATRA) on neuronal injury induced by intravitreous injection of N-methyl-d-aspartic acid (NMDA) (200nmol/eye). Treatment with ATRA for 7 days (15mg/kg for the first two days and 10mg/kg for the following five days, p.o.) reduced the decrease of cell number in the ganglion cell layer and the inner nuclear layer 7 days after NMDA injection. TUNEL staining 6h after NMDA injection showed that treatment with ATRA (15mg/kg, p.o.) 1h prior to NMDA injection reduced the number of apoptotic cells in the ganglion cell layer and inner nuclear layer. The anti-apoptotic effect of ATRA was vanished by intravitreous injection of U0126, an extracellular signal-regulated kinase/mitogen-activated protein kinase kinase inhibitor (1nmol/eye). These results suggest that ATRA has a protective effect, which is medicated by extracellular signal-regulated kinase pathway, on NMDA-induced apoptosis in the rat retina. ATRA may be useful as a therapeutic drug against retinal diseases that cause glutamate neurotoxicity.

Late preconditioning in rat retina: involvement of adenosine and ATP-sensitive K+ channel

To determine whether stimulation of adenosine receptors and opening of ATP-sensitive K(+) channels were involved in the protective effect of late preconditioning in the rat retina, rats were subjected to 60 min of retinal ischemia, and ischemic preconditioning was achieved by applying 5 min of ischemia 24 h before 60 min of ischemia. In non-preconditioned rats, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning completely prevented the retinal tissue damage and 8-phenyltheophylline or 5-hydroxydecanoate reduced the protective effect of ischemic preconditioning. Therefore, stimulation of adenosine receptors and opening of ATP-sensitive K(+) channels might be involved in the mechanism of histological protection by late preconditioning in the retina.