Can cilia provide an entry gateway for SARS-CoV-2 to human ciliated cells?

A worldwide coronavirus pandemic is in full swing and, at the time of writing, there are only few treatments that have been successful in clinical trials, but no effective antiviral treatment has been approved. Because of its lethality, it is important to understand the current strain's effects and mechanisms not only in the respiratory system but also in other affected organ systems as well. Past coronavirus outbreaks caused by SARS-CoV and MERS-CoV inflicted life-threatening acute kidney injuries (AKI) on their hosts leading to significant mortality rates, which went somewhat overlooked in the face of the severe respiratory effects. Recent evidence has emphasized renal involvement in SARS-CoV-2, stressing that kidneys are damaged in patients with COVID-19. The mechanism by which this virus inflicts AKI is still unclear, but evidence from other coronavirus strains may hold some clues. Two theories exist for the proposed mechanism of AKI: 1) the AKI is a secondary effect to reduced blood and oxygen levels causing hyperinflammation and 2) the AKI is due to cytotoxic effects. Kidneys express angiotensin-converting enzyme-2 (ACE2), the confirmed SARS-CoV-2 target receptor as well as collectrin, an ACE2 homologue that localizes to the primary cilium, an organelle historically targeted by coronaviruses. Although the available literature suggests that kidney damage is leading to higher mortality rates in patients with COVID-19, especially in those with preexisting kidney and cardiovascular diseases, the pathogenesis of COVID-19 is still being investigated. Here, we present brief literature review supporting our proposed hypothesis of a possible link between SARS-CoV-2 cellular infection and cilia.

Proteomic Identification Reveals the Role of Ciliary Extracellular-Like Vesicle in Cardiovascular Function.

Primary cilia are shown to have membrane swelling, also known as ciliary bulbs. However, the role of these structures and their physiological relevance remains unknown. Here, it is reported that a ciliary bulb has extracellular vesicle (EV)-like characteristics. The ciliary extracellular-like vesicle (cELV) has a unique dynamic movement and can be released by mechanical fluid force. To better identify the cELV, differential multidimensional proteomic analyses are performed on the cELV. A database of 172 cELV proteins is generated, and all that examined are confirmed to be in the cELV. Repressing the expression of these proteins in vitro and in vivo inhibits cELV formation. In addition to the randomized heart looping, hydrocephalus, and cystic kidney in fish, compensated heart contractility is observed in both fish and mouse models. Specifically, low circulation of cELV results in hypotension with compensated heart function, left ventricular hypertrophy, cardiac fibrosis, and arrhythmogenic characteristics, which result in a high mortality rate in mice. Furthermore, the overall ejection fraction, stroke volume, and cardiac output are significantly decreased in mice lacking cELV. It is thus proposed that the cELV as a nanocompartment within a primary cilium plays an important role in cardiovascular functions.

Signaling interplay between primary cilia and nitric oxide: A mini review.

New discoveries into the functional role of primary cilia are on the rise. In little more than 20 years, research has shown the once vestigial organelle is a signaling powerhouse involved in a vast number of essential cellular processes. In the same decade that interest in primary cilia was burgeoning, nitric oxide won molecule of the year and a Nobel prize for its role as a near ubiquitous signaling molecule. Although primary cilia and nitric oxide are both involved in signaling, a direct relationship has not been investigated; however, after a quick review of the literature, parallels between their functions can be drawn. This review aims to suggest a possible interplay between primary cilia and nitric oxide signaling especially in the areas of vascular tissue homeostasis and cellular proliferation.

Distribution and function of the muscarinic receptor subtypes in the cardiovascular system.

Muscarinic acetylcholine receptors belong to the G protein-coupled receptor superfamily and are widely known to mediate numerous functions within the central and peripheral nervous system. Thus, they have become attractive therapeutic targets for various disorders. It has long been known that the parasympathetic system, governed by acetylcholine, plays an essential role in regulating cardiovascular function. Unfortunately, due to the lack of pharmacologic selectivity for any one muscarinic receptor, there was a minimal understanding of their distribution and function within this region. However, in recent years, advancements in research have led to the generation of knockout animal models, better antibodies, and more selective ligands enabling a more thorough understanding of the unique role muscarinic receptors play in the cardiovascular system. These advances have shown muscarinic receptor 2 is no longer the only functional subtype found within the heart and muscarinic receptors 1 and 3 mediate both dilation and constriction in the vasculature. Although muscarinic receptors 4 and 5 are still not well characterized in the cardiovascular system, the recent generation of knockout animal models will hopefully generate a better understanding of their function. This mini review aims to summarize recent findings and advances of muscarinic involvement in the cardiovascular system.

Alcohol consumption impairs the ependymal cilia motility in the brain ventricles.

Ependymal cilia protrude into the central canal of the brain ventricles and spinal cord to circulate the cerebral spinal fluid (CSF). Ependymal cilia dysfunction can hinder the movement of CSF leading to an abnormal accumulation of CSF within the brain known as hydrocephalus. Although the etiology of hydrocephalus was studied before, the effects of ethanol ingestion on ependymal cilia function have not been investigated in vivo. Here, we report three distinct types of ependymal cilia, type-I, type-II and type-III classified based upon their beating frequency, their beating angle, and their distinct localization within the mouse brain-lateral ventricle. Our studies show for the first time that oral gavage of ethanol decreased the beating frequency of all three types of ependymal cilia in both the third and the lateral rat brain ventricles in vivo. Furthermore, we show for the first time that hydin, a hydrocephalus-inducing gene product whose mutation impairs ciliary motility, and polycystin-2, whose ablation is associated with hydrocephalus are colocalized to the ependymal cilia. Thus, our studies reinforce the presence of three types of ependymal cilia in the brain ventricles and demonstrate the involvement of ethanol as a risk factor for the impairment of ependymal cilia motility in the brain.

Vascular Endothelial Primary Cilia: Mechanosensation and Hypertension.

Primary cilia are sensory organelles that extend from the cell surface and sense extracellular signals. Endothelial primary cilia protruding from the inner surface of blood vessel walls sense changes in blood flow and convert this mechanosensation into an intracellular biochemical/molecular signal, which triggers a cellular response. Primary endothelial cilia dysfunction may contribute to the impairment of this response and thus be directly implicated in the development of vascular abnormalities such as hypertension and aneurysms. Using both in vitro techniques as well as in vivo animal models, we and others have investigated fluid flow mechanosensory functions of endothelial cilia in cultured cells, animal models and autosomal dominant polycystic kidney disease (ADPKD) patients. More in-depth studies directed at identification of the mechanisms of fluid flow sensing will further enhance our knowledge of cilia-dependent vascular pathology. Although the current treatments aimed at treating the cardiovascular symptoms in ADPKD patients successfully slowed the progression of cyst growth, there is growing evidence which suggests that drugs which interfere with primary cilia function or structure could reduce cardiovascular complications in ADPKD. This review is to summarize the most recent studies on primary endothelial cilia function in the vascular system and to present primary cilia as a novel therapeutic target for vascular hypertension.

Capillary endothelia from two ADPKD patients are polyploidy.

Bilateral renal cyst formation is the main feature of autosomal dominant polycystic kidney disease (ADPKD). We and other laboratories have previously shown that cyst-lining epithelia of kidneys from ADPKD patients are characterized by polyploidy. In this report, we show that endothelia from the renal capillary beds of two ADPKD patients are also polyploidy. Spectral karyotyping study further confirms our flow cytometry analyses. We suggest that polyploidy may be used as a potential cellular marker in ADPKD.

Chemical-Free Technique to Study the Ultrastructure of Primary Cilium.

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our results showed that a cilium has a swelling-like structure (termed bulb), which was previously considered a fixation artifact. The intramembrane particles observed via HPF/FFTEM indicated the presence of integral membrane proteins and soluble matrix proteins along the ciliary bulb, which is part of an integral structure within the ciliary membrane. We propose that HPF/FFTEM is an important and more suitable chemical-free method to study the ultrastructure of primary cilia.

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo's Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain's slices in a customized glass-bottom plate containing Dulbecco's Modified Eagle's Medium (DMEM)/High-Glucose at 37 °C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Protein composition and movements of membrane swellings associated with primary cilia.

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure.

Real-time vascular mechanosensation through ex vivo artery perfusion.

BACKGROUND: Cell-based perfusion studies have provided great insight into fluid-sensing mechanisms, such as primary cilia in the renal and vascular systems. However, the intrinsic limitations of in vitro cell culture, such as the inability to reflect cellular organization within tissues, has distanced observed paradigms from possible clinical developments. Here we describe a protocol that applies ex vivo artery perfusion and calcium imaging to observe real-time cellular responses to fluid-shear stress. RESULTS: Through our ex vivo artery perfusion method, we were able to simulate physiological flow and initiate distinct fluid shear stress mechanosensory responses, as well as induced acetylcholine responses in mouse aortic tissue. The observed calcium profiles confirm results found through previous in vitro cell culture experiments. The overall procedure, including dissection, sample preparation and perfusion, takes around 3 hours to complete. CONCLUSION: Through our unique method, we are able to induce laminar flow within intact mouse aortic tissue and illicit subsequent cellular responses. This method of ex vivo artery perfusion provides the opportunity to bridge the novel findings of in vitro studies with subsequent physiological models of fluid-shear stress mechanosensation in vascular tissues.

Roles of dopamine receptor on chemosensory and mechanosensory primary cilia in renal epithelial cells.

Dopamine plays a number of important physiological roles. However, activation of dopamine receptor type-5 (DR5) and its effect in renal epithelial cells have not been studied. Here, we show for the first time that DR5 is localized to primary cilia of LLCPK kidney cells. Renal epithelial cilia are mechanosensory organelles that sense and respond to tubular fluid-flow in the kidney. To determine the roles of DR5 and sensory cilia, we used dopamine to non-selectively and fenoldopam to selectively activate ciliary DR5. Compared to mock treatment, dopamine treated cells significantly increases the length of cilia. Fenoldopam further increases the length of cilia compared to dopamine treated cells. The increase in cilia length also increases the sensitivity of the cells in response to fluid-shear stress. The graded responses to dopamine- and fenoldopam-induced increase in cilia length further show that sensitivity to fluid-shear stress correlates to the length of cilia. Together, our studies suggest for the first time that dopamine or fenoldopam is an exciting agent that enhances structure and function of primary cilia. We further propose that dopaminergic agents can be used in "cilio-therapy" to treat diseases associated with abnormal cilia structure and/or function.

Survivin-induced abnormal ploidy contributes to cystic kidney and aneurysm formation.

BACKGROUND: Cystic kidneys and vascular aneurysms are clinical manifestations seen in patients with polycystic kidney disease, a cilia-associated pathology (ciliopathy). Survivin overexpression is associated with cancer, but the clinical pathology associated with survivin downregulation or knockout has never been studied before. The present studies aim to examine whether and how cilia function (Pkd1 or Pkd2) and structure (Tg737) play a role in cystic kidney and aneurysm through survivin downregulation. METHODS AND RESULTS: Cysts and aneurysms from polycystic kidney disease patients, Pkd mouse, and zebrafish models are characterized by chromosome instability and low survivin expression. This triggers cytokinesis defects and formation of nuclear polyploidy or aneuploidy. In vivo conditional mouse and zebrafish models confirm that survivin gene deletion in the kidneys results in a cystic phenotype. As in hypertensive Pkd1, Pkd2, and Tg737 models, aneurysm formation can also be induced in vascular-specific normotensive survivin mice. Survivin knockout also contributes to abnormal oriented cell division in both kidney and vasculature. Furthermore, survivin expression and ciliary localization are regulated by flow-induced cilia activation through protein kinase C, Akt and nuclear factor-κB. Circumventing ciliary function by re-expressing survivin can rescue polycystic kidney disease phenotypes. CONCLUSIONS: For the first time, our studies offer a unifying mechanism that explains both renal and vascular phenotypes in polycystic kidney disease. Although primary cilia dysfunction accounts for aneurysm formation and hypertension, hypertension itself does not cause aneurysm. Furthermore, aneurysm formation and cyst formation share a common cellular and molecular pathway involving cilia function or structure, survivin expression, cytokinesis, cell ploidy, symmetrical cell division, and tissue architecture orientation.

Non-motile primary cilia as fluid shear stress mechanosensors.

Primary cilia are sensory organelles that transmit extracellular signals into intracellular biochemical responses. Structural and functional defects in primary cilia are associated with a group of human diseases, known as ciliopathies, with phenotypes ranging from cystic kidney and obesity to blindness and mental retardation. Primary cilia mediate mechano- and chemosensation in many cell types. The mechanosensory function of the primary cilia requires the atypical G-protein-coupled receptor polycystin-1 and the calcium-permeable nonselective cation channel polycystin-2. Mechanical stimulations such as fluid-shear stress of the primary cilia initiate intracellular calcium rise, nitric oxide release, and protein modifications. In this review, we describe a set of protocols for cell culture to promote ciliation, mechanical stimulations of the primary cilia, and measurements of calcium rise and nitric oxide release induced by fluid shear stress.

Spectral karyotyping to study chromosome abnormalities in humans and mice with polycystic kidney disease.

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed (1, 2). This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities. SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope (3, 4). SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies (5, 6). SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes (7-10). The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (http://www.spectral-imaging.com/). SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia (11-13). Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models (14). Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Primary cilia regulates the directional migration and barrier integrity of endothelial cells through the modulation of hsp27 dependent actin cytoskeletal organization.

Cilia are mechanosensing organelles that communicate extracellular signals into intracellular responses. Altered functions of primary cilia play a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (Tg737(orpk/orpk) ) mouse, with impaired cilia assembly, exhibit a reduction in the actin stress fibers and focal adhesions compared to wild-type (WT). In contrast, endothelial cells from polycystin-1 deficient mice (pkd1(null/null) ), with impaired cilia function, display robust stress fibers, and focal adhesion assembly. We found that the Tg737(orpk/orpk) cells exhibit impaired directional migration and endothelial cell monolayer permeability compared to the WT and pkd1(null/null) cells. Finally, we found that the expression of heat shock protein 27 (hsp27) and the phosphorylation of focal adhesion kinase (FAK) are downregulated in the Tg737(orpk/orpk) cells and overexpression of hsp27 restored both FAK phosphorylation and cell migration. Taken together, these results demonstrate that disruption of the primary cilia structure or function compromises the endothelium through the suppression of hsp27 dependent actin organization and focal adhesion formation, which may contribute to the vascular dysfunction in ciliopathies.

Endothelial cells from humans and mice with polycystic kidney disease are characterized by polyploidy and chromosome segregation defects through survivin down-regulation.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary and systemic disorder associated with various cardiovascular complications. It has been implicated with dysfunction in primary cilia. We and others have shown that the immediate function of endothelial cilia is to sense extracellular signal. The long-term function of cilia is hypothesized to regulate cell cycle. Here, we show that ciliary function (polycystins) and structure (polaris) are required for proper cellular division. Cilia mutant cells undergo abnormal cell division with apparent defects in mitotic spindle formation, cellular spindle assembly checkpoint and centrosome amplification. Down-regulation of the chromosomal passenger survivin contributes to these abnormalities, which further result in cell polyploidy. Re-expression of survivin restores a competent spindle assembly checkpoint and reduces polyploidy. Aged animals show a more severe phenotype in cellular division, consistent with progression of cardiovascular complications seen in older ADPKD patients. For the first time, we show that structure and function of mechanosensory cilia are crucial in maintaining proper cellular proliferation. Furthermore, developmental aging plays a crucial role in the progression of these abnormal cellular phenotypes. We propose that abnormal function or structure of primary cilia not only causes failure to transmit extracellular signals, but also is associated with cytokinesis defects in both mice and humans with polycystic kidney disease.

A Comparative Study of Embedded and Anesthetized Zebrafish in vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction.

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.

Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in approximately 15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).