Adaptor protein HIP-55 promotes macrophage M1 polarization through promoting AP-1 complex activation.

Overwhelming macrophage M1 polarization induced by malfunction of the renin-angiotensin-aldosterone system (RAAS) initiates inflammatory responses, which play a crucial role in various cardiovascular diseases. However, the underlying regulatory mechanism remains elusive. Here, we identified adaptor protein HIP-55 as a critical regulator of macrophage M1 polarization. The expression of HIP-55 was upregulated in M1 macrophage induced by Ang II. Overexpression of HIP-55 significantly promoted Ang II-induced macrophage M1 polarization, whereas genetic deletion of HIP-55 inhibited the Ang II-induced macrophage M1 polarization. Mechanistically, HIP-55 facilitated activator protein-1 (AP-1) complex activation induced by Ang II via promoting ERK1/2 and JNK phosphorylation. Moreover, blocking AP-1 complex activation can attenuate the function of HIP-55 in macrophage polarization. Collectively, our results reveal the role of HIP-55 in macrophage polarization and provide potential therapeutic insights for cardiovascular diseases associated with RAAS dysfunction.

Nanoparticles Internalization through HIP-55-Dependent Clathrin Endocytosis Pathway.

Nanoparticles are promising tools for biomedicine. Many nanoparticles are internalized to function. Clathrin-mediated endocytosis is one of the most important mechanisms for nanoparticle internalization. However, the regulatory mechanism of clathrin-mediated nanoparticle endocytosis is still unclear. Here, we report that the adapter protein HIP-55 regulates clathrin-mediated nanoparticle endocytosis. CdSe/ZnS quantum dots (QDs), a typical nanoparticle, enter cells through the HIP-55-dependent clathrin endocytosis pathway. Both pharmacological inhibitor and genetic intervention demonstrate that QDs enter cells through clathrin-mediated endocytosis. HIP-55 can interact with clathrin and promote clathrin-mediated QDs endocytosis. Furthermore, HIP-55 ΔADF which is defective in F-actin binding fails to promote QDs endocytosis, indicating HIP-55 promotes clathrin-mediated QDs endocytosis depending on interaction with F-actin. In vivo, HIP-55 knockout also inhibits endocytosis of QDs. These findings reveal that HIP-55 acts as an intrinsic regulator for clathrin-mediated nanoparticle endocytosis, providing new insight into the nanoparticle internalization and a new strategy for nanodrug enrichment in target cells.

Posttranslational modifications in GPCR internalization.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors that serve as the most important drug targets. Classically, GPCR internalization has been considered to lead to receptor desensitization. However, many studies over the past decade have reported that internalized membrane receptors can trigger distinct signal activation. The "internalized activation" provides a completely new understanding for the receptor internalization, the mechanism of physiology/pathology and novel drug targets for precision medicine. GPCR internalization undergoes a series of strict regulations, especially by posttranslational modifications (PTMs). Here, this review summarizes different PTMs in GPCR internalization and analyzes their significance in GPCR internalization dynamics, internalization routes, postinternalization fates, and related diseases, which will offer new insights into the regulatory mechanism of GPCR signaling and novel drug targets for precision medicine.

The new fate of internalized membrane receptors: Internalized activation.

Classically, the fate of internalized membrane receptors includes receptor degradation and receptor recycling. However, recent findings have begun to challenge these views. Much research demonstrated that many internalized membrane receptors can trigger distinct signal activation rather than being desensitized inside the cell. Here, we introduce the concept of "internalized activation" which not only represents a new mode of receptor activation, but also endows the new fate for receptor internalization (from death to life). The new activation mode and fate of membrane receptor are ubiquitous and have unique theoretical significance. We systematically put forward the features, process, and regulation of "internalized activation" and its significance in signal transduction and diseases. "Internalized activation" will provide a completely new understanding for the theory of receptor activation, internalization and novel drug targets for precision medicine.

Internalized Activation of Membrane Receptors: From Phenomenon to Theory.

Many studies over the past decade have reported that internalized membrane receptors can trigger distinct signal activation, rather than being desensitized inside the cell. Here, we propose the concept of 'internalized activation' as a distinctive component of the receptor theory framework and exhibit its significance and role in diseases.

Angiotensin-converting enzyme 2 (ACE2): SARS-CoV-2 receptor and RAS modulator.

The coronavirus disease 2019 (COVID-19) outbreak is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Angiotensin-converting enzyme 2 (ACE2) was rapidly identified as the critical functional receptor for SARS-CoV-2. ACE2 is well-known as a counter-regulator of the renin-angiotensin system (RAS) and plays a key role in the cardiovascular system. Given that ACE2 functions as both a SARS-CoV-2 receptor and a RAS modulator, the treatment for COVID-19 presents a dilemma of how to limit virus entry but protect ACE2 physiological functions. Thus, an in-depth summary of the recent progress of ACE2 research and its relationship to the virus is urgently needed to provide possible solution to the dilemma. Here, we summarize the complexity and interplay between the coronavirus, ACE2 and RAS (including anti-RAS drugs). We propose five novel working modes for functional receptor for SARS-CoV-2 infection and the routes of ACE2-mediated virus entering host cells, as well as its regulatory mechanism. For the controversy of anti-RAS drugs application, we also give theoretical analysis and discussed for drug application. These will contribute to a deeper understanding of the complex mechanisms of underlying the relationship between the virus and ACE2, and provide guidance for virus intervention strategies.

Increased AT2R expression is induced by AT1R autoantibody via two axes, Klf-5/IRF-1 and circErbB4/miR-29a-5p, to promote VSMC migration.

Vascular remodeling can be caused by angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA), although the related mechanism remains unknown. Angiotensin II type 2 receptor (AT2R) plays multiple roles in vascular remodeling through cross-talk with AT1R in the cytoplasm. Here, we aimed to explore the role and mechanism of AT2R in AT1-AA-induced vascular smooth muscle cell (VSMC) migration, which is a key event in vascular remodeling. In vitro and in vivo, we found that AT2R can promote VSMC migration in AT1-AA-induced vascular remodeling. Moreover, AT2R expression was upregulated via Klf-5/IRF-1-mediated transcriptional and circErbB4/miR-29a-5p-mediated posttranscriptional mechanisms in response to AT1-AA. Our data provide a molecular basis for AT1-AA-induced AT2R expression by transcription factors, namely, a circular RNA and a microRNA, and showed that AT2R participated in AT1-AA-induced VSMC migration during the development of vascular remodeling. AT2R may be a potential target for the treatment of AT1-AA-induced vascular diseases.

Autoantibodies against AT1 Receptor Contribute to Vascular Aging and Endothelial Cell Senescence.

Vascular aging predisposes the elderly to the progression of many aging-related vascular disorders and leads to deterioration of cardiovascular diseases (CVD). However, the underlying mechanisms have not been clearly elucidated. Agonistic autoantibodies against angiotensin II type 1 (AT1) receptor (AT1-AAs) have been demonstrated to be pro-inflammatory and contribute to the progression of atherosclerosis. However, the association between AT1-AAs and vascular aging has not been defined. Peripheral arterial disease (PAD) is an acknowledged vascular aging-related disease. In this study, AT1-AAs were detected in the sera of patients with PAD and the positive rate was 44.44% (n=63) vs. 17.46% in non-PAD volunteers (n=63). In addition, case-control analysis showed that AT1-AAs level was positively correlated with PAD. To reveal the causal relationship between AT1-AAs and vascular aging, an AT1-AAs-positive rat model was established by active immunization. The carotid pulse wave velocity was higher, and the aortic endothelium-dependent vasodilatation was attenuated significantly in the immunized rats. Morphological staining showed thickening of the aortic wall. Histological examination showed that levels of the senescent markers were increased in the aortic tissue, mostly located at the endothelium. In addition, purified AT1-AAs-IgGs from both the immunized rats and PAD patients induced premature senescence in cultured human umbilical vein endothelial cells. These effects were significantly blocked by the AT1 receptor blocker. Taken together, our study demonstrates that AT1-AAs contribute to the progression of vascular aging and induce EC senescence through AT1 receptor. AT1-AA is a novel biomarker of vascular aging and aging-related CVD that acts to accelerate EC senescence.

Limited AT1 Receptor Internalization Is a Novel Mechanism Underlying Sustained Vasoconstriction Induced by AT1 Receptor Autoantibody From Preeclampsia.

Background Angiotensin II type 1 receptor ( AT 1R) autoantibody ( AT 1- AA ) was first identified as a causative factor in preeclampsia. Unlike physiological ligand angiotensin II (Ang II ), AT 1- AA can induce vasoconstriction in a sustained manner, causing a series of adverse effects, such as vascular injury and poor placental perfusion. However, its underlying mechanisms remain unclear. Here, from the perspective of AT 1R internalization, the present study investigated the molecular mechanism of sustained vasoconstriction induced by AT 1R autoantibody. Methods and Results In the current study, we used the vascular-ring technique to determine that AT 1- AA -positive IgG, which was obtained from the sera of preeclamptic patients, induced long-term vasoconstriction in endothelium-intact or endothelium-denuded rat thoracic arteries. The effect was caused by prolonged activation of AT 1R downstream signals in vascular smooth muscle cells, including Ca2+, protein kinase C, and extracellular signal-regulated kinase 1 and 2. Then, using subcellular protein fractionation, cell surface protein biotinylation, and total internal reflection fluorescence, we found that AT 1- AA -positive IgG resulted in significantly less AT 1R internalization than in the Ang II treatment group. Moreover, through use of fluorescent tracing and bioluminescence resonance energy transfer, we found that AT 1- AA -positive IgG cannot induce the recruitment of ß-arrestin1/2, which mediated receptor internalization. Then, the effect of sustained AT 1R activation induced by AT 1- AA -positive IgG was rescued by overexpression of ß-arrestin2. Conclusions These data suggested that limited AT 1R internalization resulting from the inhibition of ß-arrestin1/2 recruitment played an important role in sustained vasoconstriction induced by AT 1- AA -positive IgG, which may set the stage for avoiding AT 1R overactivation in the management of preeclampsia.

Active immunization using hand-push emulsification method increases the operator's risk of transcutaneous immunization.

Setting up an animal model by using active immunization methods is a common means of studying immune-related diseases or producing antibodies with high titer and high activities. However, the security during the process of pathogen emulsification remains unclear. In a physical examination, we unexpectedly noticed high levels of angiotensin II type 1 receptor autoantibody (AT1-AA) specific to the immunizing antigen in the sera of some researchers who had participated in setting up active immunization animal models, and we were puzzled about the cause of AT1-AA production. In this study, we intended to investigate whether the emulsified antigen was the source of infection in these researchers, and if so, how to prevent it from occurring. AT1-AA was detected by advanced ELISA method. The participants presented higher levels of AT1-AA compared with non-participants of the same laboratory. This phenomenon remained that some factors during the process of rat model establishment may contribute to AT1-AA production. Animal and glove penetration studies indicated the emulsified antigen infection was attributed to neither aerosol or fur touch nor penetrating through gloves. However, AT1-AA level was largely decreased in the participants after they used an automatic emulsification device. Because of the strong permeability of the adjuvant, we speculated that emulsified antigen might get access to the unprotected skin of the participants accidentally during the immunization process. These results demonstrated that accidental contacts of emulsified antigens may infect researchers during the process of traditional hand-push emulsification, resulting in high specific autoantibody levels, which can be prevented by using appropriate tools.

[Angiotensin II type 1 receptor autoantibody induces myocardial fibrosis by activating cardiac fibroblasts].

Myocardial fibrosis (MF) is an important pathological process of cardiac remodeling in patients with heart failure; however its etiology has not been clear. It has been known that the angiotensin II type 1 receptor autoantibody (AT1-AA) is present in patients with heart failure, but it is unclear whether this antibody directly causes MF. In this study, we investigated the role of AT1-AA in MF and its effects on cardiac fibroblasts (CFs). The AT1-AA positive rat model was established by active immunization method, and the measurement of indexes were made in the 8th week after active immunity. The results of heart echocardiography showed that the cardiac systolic and diastolic functions of AT1-AA positive rats were impaired with reduced left ventricular wall thickness and enlarged heart chambers. HE staining results showed that the myocardial fibers were disorganized and ruptured, and Masson staining revealed that the area of collagen fibers around the myocardium and coronary arteries was significantly increased in AT1-AA positive group compared with that of the control group (P < 0.05). Moreover, primary CFs isolated from neonatal rats were cultured and treated with AT1-AA for 48 h. CCK-8 and immunofluorescence staining results showed that AT1-AA enhanced proliferation rate of CFs (P < 0.001), and Western blot results showed that AT1-AA significantly increased expressions of collagen I (Col I), Col III, matrix metalloproteinase-2 (MMP-2) and MMP-9 in CFs (all P < 0.05). Taken together, these results suggest that AT1-AA may induce MF and cardiac dysfunction via activating CFs.

The inhibitory effect of BKCa channels induced by autoantibodies against angiotensin II type 1 receptor is independent of AT1R.

Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels. A preeclampsia model was established by passively immunizing healthy pregnant BALB/c mice with AT1-AA extracted from hybridoma culture supernatant. Blood pressure, serum AT1-AA levels, and urinary protein were measured in the immunized mice. BKCa channel expression was detected using qRT-PCR and immunohistochemical technique. The patch-clamp technique was used to record the single currents of BKCa channels in the HEK293T cells that had been transfected. AT1-AA immunized mice exhibited elevated AT1-AA and urinary protein levels compared with mice of the vehicle group. Systolic blood pressure was also increased in the immunized group. BKCa channel ß1-subunit expression was reduced in the mesenteric arteries of immunized mice. AT1-AA could inhibit the BKCa currents and the inhibitory effects were not completely reversed following the application of valsartan, an inhibitor of AT1 receptor. In conclusion, AT1-AA could decrease BKCa expression and inhibit BKCa activity independent of AT1R. These inhibitory effects are likely to be contributory factors in the promotion of increased vascular tone caused by AT1-AA in preeclampsia.

Effects of methamphetamine abuse on spatial cognitive function.

Methamphetamine (MA) abuse has been rising rapidly over the past decade, however, its impact in spatial cognitive function remains unknown. To understand its effect on visuospatial ability and spatial orientation ability, 40 MA users and 40 non-MA users conducted the Simple Reaction Task (Task 1), the Spatial Orientation Task (Task 2), and the Mental Rotation Task (Task 3), respectively. There was no significant difference in either accuracy or reaction time (RT) between 2 groups in Task 1. During Task 2, in comparison with non-MA users, MA users performed poorer on RT, but not in accuracy for foot and hand stimuli. In addition, both non-MA and MA users responded much more quickly to upward stimuli than downward stimuli on vertical surface, however, only non-MA users exhibited leftward visual field advantage in horizontal orientation processing. As for Task 3, MA users exhibited poorer performance and more errors than their healthy counterparts. For each group, linear relationship was revealed between RT and orientation angle, whereas MA abuse led to longer intercept for all stimuli involved. Our findings suggested that MA abuse may lead to a general deficit in the visuospatial ability and the spatial orientation ability with more serious impact in the former.

The mechanisms behind decreased internalization of angiotensin II type 1 receptor.

The internalization of angiotensin II type 1 receptor (AT1R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT1R, such as hypertension. However, the mechanism behind reduced AT1R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT1R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P2), which can increase the consumption of PtdIns (4,5) P2. (2) AT1R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR-ß-arrestin complex conformation. (3) The abnormal formation of vesicles or AT1R heterodimers with fewer endocytic receptors results in less AT1R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT1R recycling. In addition, lower expression of AT1R-associated protein (ATRAP) or higher expression of AT1R-associated protein 1 (ARAP1) can reduce receptor internalization.

Long-term presence of angiotensin II type 1 receptor autoantibody reduces aldosterone production by triggering Ca2+ overload in H295R cells.

Preeclamptic women are reported to have inadequate plasma volume expansion coupled with a suppressed secretion of aldosterone; however, the specific mechanism of preeclampsia remains unclear. We demonstrated that the presence of long-term angiotensin II type 1 receptor autoantibody (AT1-AA) reduces aldosterone production by triggering a Ca2+ overload in H295R cells. AT1-AA was discovered in preeclamptic women and reported to activate AT1R, and consequently elevate intracellular Ca2+. We found that AT1-AA significantly prolonged the time of intracellular Ca2+ elevation. Besides promoting aldosterone production as a second messenger, Ca2+ overload shows a cytotoxic effect. Our data reveals that long-term presence of AT1-AA triggered a Ca2+ overload and consequent impairment of aldosterone production, which could be prevented by a PKC inhibitor, Gö 6983, or a calcium channel inhibitor, nifedipine. These findings have clinical significance because AT1R blockers are not recommended for treatment of preeclampsia due to their potential harm to the fetus. Our findings also emphasize a potential clinical benefit of immunoadsorption or neutralization of AT1-AA in preeclamptic women.