Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage.

BACKGROUND AND PURPOSE: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. KEY RESULTS: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. CONCLUSION AND APPLICATIONS: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

Enhancement of excitatory transmission in NTS neurons projecting to ventral medulla of rats exposed to sustained hypoxia is blunted by minocycline.

KEY POINTS: Rats subjected to sustained hypoxia (SH) present increases in arterial pressure (AP) and in glutamatergic transmission in the nucleus tractus solitarius (NTS) neurons sending projections to ventrolateral medulla (VLM). Treatment with minocycline, a microglial inhibitor, attenuated the increase in AP in response to SH. The increase in the amplitude of glutamatergic postsynaptic currents in the NTS-VLM neurons, induced by postsynaptic mechanisms, was blunted by minocycline treatment. The number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS of minocycline-treated rats. The data show that microglial recruitment/proliferation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the observed increase in AP. ABSTRACT: Short-term sustained hypoxia (SH) produces significant autonomic and respiratory adjustments and triggers activation of microglia, the resident immune cells in the brain. SH also enhances glutamatergic neurotransmission in the NTS. Here we evaluated the role of microglial activation induced by SH on the cardiovascular changes and mainly on glutamatergic neurotransmission in NTS neurons sending projections to the ventrolateral medulla (NTS-VLM), using a microglia inhibitor (minocycline). Direct measurement of arterial pressure (AP) in freely moving rats showed that SH (24 h, fraction of inspired oxygen ( FI,O2 ) 0.1) in vehicle and minocycline (30 mg/kg i.p. for 3 days)-treated groups produced a significant increase in AP in relation to control groups under normoxic conditions, but this increase was significantly lower in minocycline-treated rats. Whole-cell patch-clamp recordings revealed that the active properties of the membrane were comparable among the groups. Nevertheless, the amplitudes of glutamatergic postsynaptic currents, evoked by tractus solitarius stimulation, were increased in NTS-VLM neurons of SH rats. Changes in asynchronous glutamatergic currents indicated that the observed increase in amplitude was due to postsynaptic mechanisms. These changes were blunted in the SH group previously treated with minocycline. Using immunofluorescence, we found that the number of microglial cells was increased in the NTS of vehicle-treated SH rats but not in the NTS neurons of minocycline-treated rats. Our data support the concept that microglial activation induced by SH is associated with the enhancement of excitatory neurotransmission in NTS-VLM neurons, which may contribute to the increase in AP observed in this experimental model.

Intrinsic properties of rostral ventrolateral medulla presympathetic and bulbospinal respiratory neurons of juvenile rats are not affected by chronic intermittent hypoxia.

The presympathetic neurons in the rostral ventrolateral medulla (RVLM) are considered to be the source of the sympathetic activity, and there is experimental evidence that these cells present intrinsic autodepolarization. There is also evidence that an important respiratory neuronal population located in the RVLM/Bötzinger complex (BötC) corresponds to augmenting expiratory neurons (aug-E), which send projections to the phrenic nucleus in the spinal cord. However, the pacemaker activity of presympathetic neurons and the intrinsic properties of aug-E neurons had not been evaluated in brainstem slices of juvenile rats (postnatal day 35). Chronic intermittent hypoxia (CIH) is a sympathetic-mediated hypertension model, which seems to produce an associated increase in the activity of aug-E neurons. In this study, we evaluated the effects of CIH on the intrinsic properties of RVLM/BötC presympathetic and phrenic nucleus-projecting neurons (aug-E) in brainstem slices of juvenile rats (postnatal day 35). We observed that all presympathetic neurons presented spontaneous action potential firing (n = 18), which was not abolished by ionotropic receptor antagonism. In addition, exposure to 10 days of CIH produced no changes in their intrinsic passive properties, firing pattern or excitability. Most aug-E neurons presented spontaneous firing in control conditions (13 of 15 neurons), and this characteristic was preserved after blocking fast synaptic transmission (12 of 15 neurons), clearly demonstrating their intrinsic pacemaker activity. Chronic intermittent hypoxia also produced no changes in intrinsic passive properties, frequency and pattern of discharge or excitability of the aug-E neurons. The present study shows that: (i) it is possible to record the electrophysiological properties of RVLM/BötC presympathetic and aug-E neurons in brainstem slices from juvenile rats; (ii) these neurons present characteristics of intrinsic pacemakers; and (iii) their intrinsic properties were not altered by chronic intermittent hypoxia.

Chronic intermittent hypoxia depresses afferent neurotransmission in NTS neurons by a reduction in the number of active synapses.

Long-term synaptic plasticity has been recently described in brainstem areas associated to visceral afferent sensory integration. Chronic intermittent hypoxia (CIH), an animal model for studying obstructive sleep apnea in humans, depresses the afferent neurotransmission in nucleus tractus solitarii (NTS) neurons, which affect respiratory and autonomic regulation. Here we identified the synaptic mechanisms of CIH-induced depression of the afferent neurotransmission in NTS neurons in juvenile rats. We verified that CIH reduced the amplitude of both NMDA and non-NMDA glutamatergic excitatory currents (eEPSCs) evoked by tractus solitarii stimulation (TS-eEPSC) of second-order neurons in the NTS. No changes were observed in release probability, evidenced by absence of any CIH-elicited effects on short-term depression and failures in EPSCs evoked in low calcium. CIH also produced no changes in TS-eEPSC quantal size, since the amplitudes of both low calcium-evoked EPSCs and asynchronous TS-eEPSCs (evoked in the presence of Sr(2+)) were unchanged. Using single TS afferent fiber stimulation in slices from control and CIH rats we clearly show that CIH reduced the quantal content of the TS-eEPSCs without affecting the quantal size or release probability, suggesting a reduction in the number of active synapses as the mechanism of CIH induced TS-eEPSC depression. In accordance with this concept, the input-output relationship of stimulus intensity and TS-eEPSC amplitude shows an early saturation in CIH animals. These findings open new perspectives for a better understanding of the mechanisms underlying the synaptic plasticity in the brainstem sensory neurons under challenges such as those produced by CIH in experimental and pathological conditions.

Controle neural da circulação e hipertensão arterial / Central neural control of the circulation and high blood pressure

Existem várias evidências experimentais mostrando a participação do sistema nervoso central (SNC) o controle autonômico da função cardiovascular. No entanto,os mecanismos neuroquímicos envolvidos nesses diferentes circuitos neuronais ainda não estão totalmente esclarecidos. Nessa revisão, apresentamos aspectos dos mecanismos neurais envolvidos no controle da circulação, com foco nas estruturas bulbares responsáveis na geração e modulação da atividade autonômica simpática, bem como nas respostas cardiovasculares reflexas produzidas pela ativação do barorreflexo. Estudos neuroanatômicos e funcionais mostraram que o NTS é a área no tronco cerebral na qual o barorreflexo faz sua primeira sinapse, sendo também a área de integração desse reflexo, a qual por meio de conexões com outras estruturas bulbares promove a modulação das atividades eferentes autonômicas para sistema cardiovascular regulando, desse modo, os níveis da PA. A estimulação do barorreflexo promove inibição da via simpática, por meio da ativação das projeções do NTS para o CVLM e desse para o RVLM. Quando ativados, os neurônios do CVLM exercem uma ação inibitória sobre os neurônios do RVLM, os quais são responsáveis pela geração da atividade eferente simpática, diminuindo assim a freqüência de despolarização desses neurônios e, conseqüentemente, os níveis da PA. Outras áreas do SNC, como o núcleo PVN, e alguns neuromoduladores, como a Ang II e o NO, podem também influenciar a atividade dos neurônios do RVLM e a atividade eferente simpática. Alterações nos diferentes níveis do sistema neural de regulação cardiovascular podem resultar em hiperatividade simpática e conseqüentemente em hipertensão arterial

Respiratory and autonomic responses to microinjection of NMDA and AMPA into the commissural subnucleus of the NTS of awake rats.

The changes in mean arterial pressure (MAP) and respiratory frequency (RF) in response to microinjection of NMDA or AMPA into the commissural subnucleus of the NTS (comNTS) at the calamus scriptorius level of awake rats were evaluated. Under tribromoethanol anesthesia, the rats received guide-cannulae in direction of the NTS and a catheter was inserted into the femoral artery for measurement of arterial pressure. Changes in RF were evaluated with the rats inside a plethysmographic chamber. Randomly microinjections of 5 doses of NMDA (0.001, 0.01, 0.1, 1.0 and 2 nmol/50 nL; n = 10) or AMPA (1, 5, 10, 25 and 50 pmol/50 nL; n = 8) into the comNTS were performed at 15 min intervals and produced a dose-dependent increase in MAP [NMDA (3 +/- 2, 4 +/- 3, 25 +/- 4, 41 +/- 4 and 51 +/- 4 mm Hg) and AMPA (0 +/- 1, 14 +/- 4, 17 +/- 3, 27 +/- 5 and 34 +/- 3 mm Hg)]. Microinjection of NMDA (1 nmol/50 nL; n = 7) or AMPA (50 pmol/50 nL; n = 4) into the comNTS produced a long lasting apnea. The pressor responses to microinjection of NMDA or AMPA into the comNTS were blocked by prazosin, a alpha(1)-adrenoceptor antagonist, indicating that the increase in arterial pressure in both cases was sympathetically mediated. The data show that microinjection of NMDA and AMPA into the comNTS produced pressor response and apnea, indicating that both ionotropic l-glutamate receptors may play a role in the neurotransmission/neuromodulation of the autonomic and respiratory components of the cardiovascular reflexes at this level.