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A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke <em>In Vitro</em>

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

University of Miami

The blood-brain barrier (BBB) is essential for the homeostasis of the central nervous system (CNS). Functions of the BBB are performed by the neurovascular unit (NVU), which consists of endothelial cells, pericytes, astrocytes, microglia, basement membrane, and neurons. NVU cells interact closely and together are responsible for neurovascular coupling, BBB integrity, and transendothelial fluid transport. Studies have shown that NVU dysfunction is implicated in several acute and chronic neurological diseases, including Alzheimer's disease, multiple sclerosis, and stroke. The mechanisms of NVU disruption remain poorly understood, partially due to difficulties in selective targeting of NVU cells. In this review, we discuss the relative merits of available protein markers and drivers of the NVU along with recent advancements that have been made in the field to increase efficiency and specificity of NVU research.

In Vivo Targeting of the Neurovascular Unit: Challenges and Advancements.

Urinary incontinence is an involuntary urination (leakage of urine). About 200 million people suffer from this condition, and 60% of cases are concealed and untreated because of shame. It is estimated that an increasing number of young women and women of menopausal age will suffer from urinary incontinence. This disease occurs during the perinatal, perimenopausal period, as a result of brain damage or an unhealthy lifestyle. There are four main types of urinary incontinence: stress, urge, overflow and mixed form. Treatment is adapted to the severity of disease, its type and includes physiotherapeutic treatment (kinesiotherapy, physiotherapy, massage), pharmacological, psychological and surgical treatment. In recent years, growing interest has been observed in the noninvasive biofeedback method. The patient learns to contract the weakened pelvic floor muscles, constantly monitoring progress in treatment. She is also motivated by visual and auditory stimuli. Growing evidence confirms the effectiveness of this method, which to a large extent eliminates urinary incontinence. Nevertheless, attention should also be paid to prevention, which reduces the risk of involuntary leakage of urine.

Urinary incontinence in women: biofeedback as an innovative treatment method.

HIV-1 enters the brain by altering properties of the blood-brain barrier (BBB). Recent evidence indicates that among cells of the BBB, pericytes are prone to HIV-1 infection. Occludin (ocln) and caveolin-1 (cav-1) are critical determinants of BBB integrity that can regulate barrier properties of the BBB in response to HIV-1 infection. Additionally, Alix is an early acting endosomal factor involved in HIV-1 budding from the cells. The aim of the present study was to evaluate the role of cav-1, ocln, and Alix in HIV-1 infection of brain pericytes. Our results indicated that cav-1, ocln, and Alix form a multi-protein complex in which they cross-regulate each other's expression. Importantly, the stability of this complex was affected by HIV-1 infection. Modifications of the complex resulted in diminished HIV-1 infection and alterations of the cytokine profile produced by brain pericytes. These results identify a novel mechanism involved in HIV-1 infection contributing to a better understanding of the HIV-1 pathology and the associated neuroinflammatory responses.

Occludin, caveolin-1, and Alix form a multi-protein complex and regulate HIV-1 infection of brain pericytes.

 Neurological complications are common in patients affected by COVID-19 due to the ability of SARS-CoV-2 to infect brains. While the mechanisms of this process are not fully understood, it has been proposed that SARS-CoV-2 can infect the cells of the neurovascular units (NVU), which form the blood-brain barrier (BBB). The aim of the current study was to analyze the expression pattern of the main SARS-CoV-2 receptors in naïve and HIV-1-infected cells of the NVU in order to elucidate a possible pathway of the virus entry into the brain and a potential modulatory impact of HIV-1 in this process.  The gene and protein expression profile of ACE2, TMPRSS2, ADAM17, BSG, DPP4, AGTR2, ANPEP, cathepsin B and cathepsin L was assessed by qPCR and immunoblotting, respectively. In addition, we investigated if brain endothelial cells can be affected by the exposure to the S1 subunit of the S protein, the domain responsible for the direct binding of SARS-CoV-2 to the ACE2 receptors.  The receptors involved in SARS-CoV-2 infection are coexpressed in the cells of the NVU, especially in astrocytes and microglial cells. These receptors are functionally active as exposure of endothelial cells to the SARS CoV-2 S1 protein subunit altered the expression pattern of tight junction proteins, such as claudin-5 and ZO-1. Additionally, HIV-1 infection upregulated ACE2 and TMPRSS2 expression in brain astrocytes and microglia cells.  These findings provide key insight into SARS-CoV-2 recognition by cells of the NVU and may help to develop possible treatment of CNS complications of COVID-19.

Expression of SARS-CoV-2-related Receptors in Cells of the Neurovascular Unit: Implications for HIV-1 Infection.

Neurological complications are common in patients affected by COVID-19 due to the ability of SARS-CoV-2 to infect brains. While the mechanisms of this process are not fully understood, it has been proposed that SARS-CoV-2 can infect the cells of the neurovascular unit (NVU), which form the blood-brain barrier (BBB). The aim of the current study was to analyze the expression pattern of the main SARS-CoV-2 receptors in naïve and HIV-1-infected cells of the NVU in order to elucidate a possible pathway of the virus entry into the brain and a potential modulatory impact of HIV-1 in this process.

Expression of SARS-CoV-2-related receptors in cells of the neurovascular unit: implications for HIV-1 infection.

The neurovascular units (NVU) are the minimal functional units of the blood-brain barrier (BBB), composed of endothelial cells, pericytes, astrocytes, microglia, neurons, and the basement membrane. The BBB serves as an important interface for immune communication between the brain and peripheral circulation. Disruption of the NVU by the human immunodeficiency virus-1 (HIV-1) induces dysfunction of the BBB and triggers inflammatory responses, which can lead to the development of neurocognitive impairments collectively known as HIV-1-associated neurocognitive disorders (HAND). Methamphetamine (METH) use disorder is a frequent comorbidity among individuals infected with HIV-1. METH use may be associated not only with rapid HIV-1 disease progression but also with accelerated onset and increased severity of HAND. However, the molecular mechanisms of METH-induced neuronal injury and cognitive impairment in the context of HIV-1 infection are poorly understood. In this review, we summarize recent progress in the signaling pathways mediating synergistic impairment of the BBB and neuronal injury induced by METH and HIV-1, potentially accelerating the onset or severity of HAND in HIV-1-positive METH abusers. We also discuss potential therapies to limit neuroinflammation and NVU damage in HIV-1-infected METH abusers.

Synergistic Impairment of the Neurovascular Unit by HIV-1 Infection and Methamphetamine Use: Implications for HIV-1-Associated Neurocognitive Disorders.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs)  infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs)  infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non- productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

COVID-19, which is caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), has resulted in devastating morbidity and mortality worldwide due to lethal pneumonia and respiratory distress. In addition, the central nervous system (CNS) is well documented to be a target of SARS-CoV-2, and studies detected SARS-CoV-2 in the brain and the cerebrospinal fluid of COVID-19 patients. The blood-brain barrier (BBB) was suggested to be the major route of SARS-CoV-2 infection of the brain. Functionally, the BBB is created by an interactome between endothelial cells, pericytes, astrocytes, microglia, and neurons, which form the neurovascular units (NVU). However, at present, the interactions of SARS-CoV-2 with the NVU and the outcomes of this process are largely unknown. Moreover, age was described as one of the most prominent risk factors for hospitalization and deaths, along with other comorbidities such as diabetes and co-infections. This review will discuss the impact of SARS-CoV-2 on the NVU, the expression profile of SARS-CoV-2 receptors in the different cell types of the CNS and the possible role of aging in the neurological outcomes of COVID-19. A special emphasis will be placed on mitochondrial functions because dysfunctional mitochondria are also a strong inducer of inflammatory reactions and the "cytokine storm" associated with SARS-CoV-2 infection. Finally, we will discuss possible drug therapies to treat neural endothelial function in aged patients, and, thus, alleviate the neurological symptoms associated with COVID-19.

Article	Year
In Vivo Targeting of the Neurovascular Unit: Challenges and Advancements. Cellular and molecular neurobiology\| PubMed ID: 34086179	2022
Urinary incontinence in women: biofeedback as an innovative treatment method. Therapeutic advances in urology\| PubMed ID: 32647538	2020
Occludin, caveolin-1, and Alix form a multi-protein complex and regulate HIV-1 infection of brain pericytes. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 33058236	2020
Expression of SARS-CoV-2-related Receptors in Cells of the Neurovascular Unit: Implications for HIV-1 Infection. Research square\| PubMed ID: 33655239	2021
Expression of SARS-CoV-2-related receptors in cells of the neurovascular unit: implications for HIV-1 infection. Journal of neuroinflammation\| PubMed ID: 34325716	2021
Synergistic Impairment of the Neurovascular Unit by HIV-1 Infection and Methamphetamine Use: Implications for HIV-1-Associated Neurocognitive Disorders. Viruses\| PubMed ID: 34578464	2021
SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling. bioRxiv : the preprint server for biology\| PubMed ID: 35734080	2022
SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling. Research square\| PubMed ID: 35734086	2022
Role of aging in Blood-Brain Barrier dysfunction and susceptibility to SARS-CoV-2 infection: impacts on neurological symptoms of COVID-19. Fluids and barriers of the CNS\| PubMed ID: 35982454	2022

Silvia Torices

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