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New findings What is the central question of this study? Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance? Downregulation of AFAP1-AS1 blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via the miR-195/E2F3 axis, indicating thatAFAP1-AS might be a potential biomarker for HSCR patients. Abstract Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity of AFAP1-AS1 for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction of AFAP1-AS1 and miR-195 and of miR-195 and E2F transcription factor 3 (E2F3). AFAP1-AS1 was reduced in HSCR patients. Meanwhile, knockdown of AFAP1-AS1 reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest. E2F3 was diminished when miR-195 was upregulated, and AFAP1-AS1 inhibition reduced its ability to bind to miR-195. Altogether, AFAP1-AS1 silencing acts as an endogenous RNA by interacting with miR-195 to alter E2F3 expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.

An enhancement of the cardiac sympathetic afferent reflex (CSAR) contributes to sympathetic activation in renovascular hypertension. Angiotensin II in the paraventricular nucleus (PVN) augments the CSAR and increases sympathetic outflow and blood pressure. The present study aimed to determine whether endogenous hydrogen peroxide in the PVN mediated the enhanced CSAR, sympathetic activity and the effects of angiotensin II in the PVN in renovascular hypertension induced by the two-kidney, one-clip method (2K1C) in rats. At the end of the fourth week, the rats underwent sino-aortic and vagal denervation under general anaesthesia with urethane and α-chloralose. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. The CSAR was evaluated by the RSNA response to epicardial application of bradykinin. Microinjection of polyethylene glycol–catalase (PEG-CAT), an analogue of endogenous catalase, into the PVN decreased the RSNA and MAP and abolished the CSAR in both sham-operated and 2K1C rats. Microinjection into the PVN of the catalase inhibitor, aminotriazole, increased the RSNA and MAP and enhanced the CSAR. The effects of PEG-CAT or aminotriazole were greater in 2K1C rats than in sham-operated animals. The effects of angiotensin II in the PVN were abolished by pretreatment with PEG-CAT in both sham-operated and 2K1C rats; however, aminotriazole failed to potentiate the effects of angiotensin II. The catalase activity was decreased but the H2O2 levels were increased in the PVN of 2K1C rats. These results indicate that endogenous H2O2 in the PVN not only mediates the enhanced sympathetic activity and CSAR, but also the effects of angiotensin II in the PVN in renovascular hypertensive rats.

Stimulation of cardiac sympathetic afferents increases sympathetic outflow and blood pressure. Chemicals released during myocardial ischaemia activate cardiac afferents. This study was to determine the responses of neurons in paraventricular nucleus (PVN) to the cardiac afferent activation caused by exogenous chemicals or myocardial ischaemia using an extracellular single-unit recording method. Rats were anaesthetized and underwent bilateral cervical vagal denervation (VD) and carotid and aortic baroreceptor denervation (BD). In 196 spontaneously active neurons in parvicellular PVN, 60 (30.6%), 36 (18.4%) and 91 (46.4%) neurons were respectively sensitive, mildly sensitive and insensitive to capsaicin, while nine (4.6%) neurons showed inhibitory responses to capsaicin. Epicardial application of capsaicin activated capsaicin-sensitive neurons in the PVN and increased mean arterial pressure. These neurons were also sensitive to exogenous bradykinin, adenosine and H2O2. The neuron response is not secondary to a capsaicin-induced increase in mean arterial pressure because a similar degree of pressor response induced by aortic coarctation did not increase the neuron activity. Compared with intact rats, VD or BD or combined VD and BD increased the response of capsaicin-sensitive neurons to epicardial application of capsaicin, while stimulation of vagal afferents inhibited the response. Myocardial ischaemia caused increases in the activity of capsaicin-sensitive neurons and renal sympathetic nerve activity. The results indicate that chemical stimulation of cardiac sympathetic afferents activates capsaicin-sensitive neurons in parvicellular PVN, which is inhibited by the afferent activities of vagi and arterial baroreceptors. Acute myocardial ischaemia activates capsaicin-sensitive neurons in PVN and enhances sympathetic outflow.

Sympathetic activity is enhanced in hypertension, which contributes to the pathogenesis of hypertension and progression of organ damage. The cardiac sympathetic afferent reflex (CSAR) is enhanced in renovascular hypertension and involved in the sympathetic activation. The present study was designed to investigate whether angiotensin II (Ang II) and Ang II type 1 (AT(1)) receptors in paraventricular nucleus (PVN) contribute to the enhanced CSAR and sympathetic outflow in experimental renovascular hypertensive rats. Hypertension was induced by the two-kidney one-clip (2K1C) method. The normotensive rats underwent sham operation (Sham). Acute experimentation was carried out at the end of the 4th week. Under urethane and α-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in rats with sino-aortic denervation and cervical vagotomy. The AT(1) receptor expression was determined with Western blot. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. The AT(1) receptor expression in the PVN was increased, and Ang II in the PVN augmented the enhanced CSAR and RSNA in 2K1C rats. The effects of Ang II were abolished by pretreatment with the AT(1) receptor antagonist, losartan, in 2K1C rats. Losartan in the PVN normalized the enhanced CSAR and decreased the RSNA and MAP in 2K1C rats. These results indicate that the increased activity of AT(1) receptors in the PVN contributes to the enhanced CSAR and excessive sympathetic activation in renovascular hypertensive rats.

Sympathetic outflow is increased in hypertension. The aim of the present study was to investigate whether the cardiac sympathetic afferent reflex (CSAR) is enhanced in two-kidney one-clip (2K1C) renovascular hypertensive rats, and whether the enhanced CSAR contributes, in part, to the increased sympathetic outflow. Furthermore, the role of central angiotensin II type 1 (AT1) receptors in mediating the CSAR was determined. Under urethane and α-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic denervated and cervical vagotomized rats. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. Compared with sham-operated rats, the CSAR, baseline RSNA and plasma noradrenaline level were significantly enhanced in 2K1C rats. Intrapericardial administration of resiniferatoxin, which abolishes the CSAR because of the desensitization of transient receptor potential vanilloid 1-containing cardiac afferent fibres, decreased the RSNA and MAP. The enhanced CSAR in 2K1C rats was normalized by intracerebroventricular administration of the AT1 receptor antagonist losartan. Intracerebroventricular administration of angiotensin II further potentiated the enhanced CSAR in 2K1C rats, a response which was abolished by pretreatment with losartan. These results indicate that the CSAR is enhanced in 2K1C rats and the enhanced CSAR contributes, in part, to the sympathetic activation and hypertension. Central AT1 receptors are involved in the enhanced CSAR in 2K1C rats.

Our previous studies have shown that angiotensin II and reactive oxygen species in the paraventricular nucleus (PVN) modulate the cardiac sympathetic afferent reflex (CSAR). The present study was designed to demonstrate more conclusively that the PVN is an important component of the central neurocircuitry of the CSAR. In anaesthetized Sprague-Dawley rats with sinoaortic denervation and cervical vagotomy, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. The CSAR was evaluated by the response of the RSNA to epicardial application of bradykinin or capsaicin. Bilateral microinjection of the anaesthetic, lignocaine, into the PVN abolished the CSAR without significant effects on the baseline RSNA and MAP, while l-glutamate, which excites the neurons in the PVN, enhanced the CSAR and increased the baseline RSNA and MAP. Bilateral electrolytic lesions of the PVN irreversibly abolished the CSAR without significant effects on the baseline RSNA and MAP. Bilateral selective lesions of the neurons in the PVN with kainic acid induced rapid and great increases in both RSNA and MAP which returned to nearly normal levels in 60 min. At the 90th minute after kainic acid, epicardial application of bradykinin or capsaicin failed to induce the CSAR. These results indicate that inhibition or lesion of the PVN abolishes the CSAR, but excitation of the neurons in the PVN enhances the CSAR, suggesting that the PVN is an important component of the central neurocircuitry of the CSAR.

An enhancement of the cardiac sympathetic afferent reflex (CSAR) contributes to sympathetic activation in renovascular hypertension. Angiotensin II in the paraventricular nucleus (PVN) augments the CSAR and increases sympathetic outflow and blood pressure. The present study aimed to determine whether endogenous hydrogen peroxide in the PVN mediated the enhanced CSAR, sympathetic activity and the effects of angiotensin II in the PVN in renovascular hypertension induced by the two-kidney, one-clip method (2K1C) in rats. At the end of the fourth week, the rats underwent sino-aortic and vagal denervation under general anaesthesia with urethane and α-chloralose. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. The CSAR was evaluated by the RSNA response to epicardial application of bradykinin. Microinjection of polyethylene glycol-catalase (PEG-CAT), an analogue of endogenous catalase, into the PVN decreased the RSNA and MAP and abolished the CSAR in both sham-operated and 2K1C rats. Microinjection into the PVN of the catalase inhibitor, aminotriazole, increased the RSNA and MAP and enhanced the CSAR. The effects of PEG-CAT or aminotriazole were greater in 2K1C rats than in sham-operated animals. The effects of angiotensin II in the PVN were abolished by pretreatment with PEG-CAT in both sham-operated and 2K1C rats; however, aminotriazole failed to potentiate the effects of angiotensin II. The catalase activity was decreased but the H(2)O(2) levels were increased in the PVN of 2K1C rats. These results indicate that endogenous H(2)O(2) in the PVN not only mediates the enhanced sympathetic activity and CSAR, but also the effects of angiotensin II in the PVN in renovascular hypertensive rats.

Stimulation of cardiac sympathetic afferents increases sympathetic outflow and blood pressure. Chemicals released during myocardial ischaemia activate cardiac afferents. This study was to determine the responses of neurons in paraventricular nucleus (PVN) to the cardiac afferent activation caused by exogenous chemicals or myocardial ischaemia using an extracellular single-unit recording method. Rats were anaesthetized and underwent bilateral cervical vagal denervation (VD) and carotid and aortic baroreceptor denervation (BD). In 196 spontaneously active neurons in parvicellular PVN, 60 (30.6%), 36 (18.4%) and 91 (46.4%) neurons were respectively sensitive, mildly sensitive and insensitive to capsaicin, while nine (4.6%) neurons showed inhibitory responses to capsaicin. Epicardial application of capsaicin activated capsaicin-sensitive neurons in the PVN and increased mean arterial pressure. These neurons were also sensitive to exogenous bradykinin, adenosine and H(2)O(2). The neuron response is not secondary to a capsaicin-induced increase in mean arterial pressure because a similar degree of pressor response induced by aortic coarctation did not increase the neuron activity. Compared with intact rats, VD or BD or combined VD and BD increased the response of capsaicin-sensitive neurons to epicardial application of capsaicin, while stimulation of vagal afferents inhibited the response. Myocardial ischaemia caused increases in the activity of capsaicin-sensitive neurons and renal sympathetic nerve activity. The results indicate that chemical stimulation of cardiac sympathetic afferents activates capsaicin-sensitive neurons in parvicellular PVN, which is inhibited by the afferent activities of vagi and arterial baroreceptors. Acute myocardial ischaemia activates capsaicin-sensitive neurons in PVN and enhances sympathetic outflow.

Sympathetic activity is enhanced in hypertension, which contributes to the pathogenesis of hypertension and progression of organ damage. The cardiac sympathetic afferent reflex (CSAR) is enhanced in renovascular hypertension and involved in the sympathetic activation. The present study was designed to investigate whether angiotensin II (Ang II) and Ang II type 1 (AT(1)) receptors in paraventricular nucleus (PVN) contribute to the enhanced CSAR and sympathetic outflow in experimental renovascular hypertensive rats. Hypertension was induced by the two-kidney one-clip (2K1C) method. The normotensive rats underwent sham operation (Sham). Acute experimentation was carried out at the end of the 4th week. Under urethane and α-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in rats with sino-aortic denervation and cervical vagotomy. The AT(1) receptor expression was determined with Western blot. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. The AT(1) receptor expression in the PVN was increased, and Ang II in the PVN augmented the enhanced CSAR and RSNA in 2K1C rats. The effects of Ang II were abolished by pretreatment with the AT(1) receptor antagonist, losartan, in 2K1C rats. Losartan in the PVN normalized the enhanced CSAR and decreased the RSNA and MAP in 2K1C rats. These results indicate that the increased activity of AT(1) receptors in the PVN contributes to the enhanced CSAR and excessive sympathetic activation in renovascular hypertensive rats.

Sympathetic outflow is increased in hypertension. The aim of the present study was to investigate whether the cardiac sympathetic afferent reflex (CSAR) is enhanced in two-kidney one-clip (2K1C) renovascular hypertensive rats, and whether the enhanced CSAR contributes, in part, to the increased sympathetic outflow. Furthermore, the role of central angiotensin II type 1 (AT(1)) receptors in mediating the CSAR was determined. Under urethane and alpha-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in sinoaortic denervated and cervical vagotomized rats. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. Compared with sham-operated rats, the CSAR, baseline RSNA and plasma noradrenaline level were significantly enhanced in 2K1C rats. Intrapericardial administration of resiniferatoxin, which abolishes the CSAR because of the desensitization of transient receptor potential vanilloid 1-containing cardiac afferent fibres, decreased the RSNA and MAP. The enhanced CSAR in 2K1C rats was normalized by intracerebroventricular administration of the AT(1) receptor antagonist losartan. Intracerebroventricular administration of angiotensin II further potentiated the enhanced CSAR in 2K1C rats, a response which was abolished by pretreatment with losartan. These results indicate that the CSAR is enhanced in 2K1C rats and the enhanced CSAR contributes, in part, to the sympathetic activation and hypertension. Central AT(1) receptors are involved in the enhanced CSAR in 2K1C rats.

Our previous studies have shown that angiotensin II and reactive oxygen species in the paraventricular nucleus (PVN) modulate the cardiac sympathetic afferent reflex (CSAR). The present study was designed to demonstrate more conclusively that the PVN is an important component of the central neurocircuitry of the CSAR. In anaesthetized Sprague-Dawley rats with sinoaortic denervation and cervical vagotomy, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. The CSAR was evaluated by the response of the RSNA to epicardial application of bradykinin or capsaicin. Bilateral microinjection of the anaesthetic, lignocaine, into the PVN abolished the CSAR without significant effects on the baseline RSNA and MAP, while l-glutamate, which excites the neurons in the PVN, enhanced the CSAR and increased the baseline RSNA and MAP. Bilateral electrolytic lesions of the PVN irreversibly abolished the CSAR without significant effects on the baseline RSNA and MAP. Bilateral selective lesions of the neurons in the PVN with kainic acid induced rapid and great increases in both RSNA and MAP which returned to nearly normal levels in 60 min. At the 90th minute after kainic acid, epicardial application of bradykinin or capsaicin failed to induce the CSAR. These results indicate that inhibition or lesion of the PVN abolishes the CSAR, but excitation of the neurons in the PVN enhances the CSAR, suggesting that the PVN is an important component of the central neurocircuitry of the CSAR.