Primary cilium is an antenna-like and non-motile structure protruding from the apical surface of most mammalian cells including endothelial cells lining the inner side of all the blood vessels in our body. Although it has been over a century since primary cilia were discovered, the investigation about their mechano-sensing and other roles in maintaining normal functions of cardiovascular system has just started in recent years. This focused review aims to give an update about the current literature for the role of endothelial primary cilia in blood flow mechano-sensing and shear stress-shielding. To do this, we first summarized the characteristic features of endothelial primary cilia in terms of structure, dimension, molecular composition, and mechanical properties (e.g., bending rigidity), which are the dominant factors for their functions in mechano-sensing and transduction, as well as vascular protection from the blood flow-induced wall shear stress. We also described the experimental techniques and mathematical models for determining the dimension and mechanical properties of the primary cilium. Then we reviewed the molecular mechanisms underlying mechano-sensing and transduction by endothelial primary cilia and the mathematical model prediction for their roles in redistribution and reduction of wall shear stresses. Finally, we briefly discussed the common cardiovascular diseases, e.g., atherosclerosis, hypertension, and aneurysm, due to defects and malfunction of endothelial primary cilia and suggested potential targets for therapeutic treatments.
Primary cilia are microtubule-formed organelles discovered in many types of mammalian cells including epithelial cells (
Vascular endothelial cells (ECs) lining the inner wall of our blood vessels are continuously exposed to the blood flow. In order to maintain proper functions of the cardiovascular system, ECs should have a variety of mechano-sensors and transducers to sense the blood flow change and adjust the blood perfusion rate (e.g., through vessel diameter) and transport across the vessel wall accordingly. So far, at least a dozen EC mechano-sensors and transducers have been identified either on the EC surface, or at the intra- and trans-EC membrane, or within the EC cytoskeleton. Several prominent ones are endothelial surface glycocalyx, caveolae, integrins, VE-cadherins, PECAM-1, G-protein-coupled receptors and G-proteins, actin filaments, nesprins, and primary cilia (
The antenna-like primary cilium is considered as a cellular organelle, which mainly consists of a membrane, soluble compartment (cilioplasm), axoneme, basal body and ciliary tip. The structure of a general primary cilium is depicted in
Various molecules residing on the primary cilia contribute to the maintenance of the proper ciliary structure and function. Any alteration in the fluid pressure and shear stress can be detected by the sensory proteins localized on the cilia and these alterations are transduced into the interior of the cell via various signaling pathways. The signaling pathways organized by primary cilia are rather diverse and depend on the cell type. Here we only focus on several proteins that are widely described on endothelial primary cilia, which are illustrated in
Intraflagellar transport (IFT) particles, including intraflagellar transport protein 88 (IFT88), are essential for building primary cilia, or ciliogenesis. During ciliogenesis, the centrosome migrates towards the cell surface when a cell enters G0 (resting state) and the mother centriole attaches to a Golgi-derived vesicle (
Polycystin-1 and -2 (PC1/2) have been thought to form a heteromeric ion channel complex on the primary cilia, regulating Ca2+ influx into the cell (
The TRP channels (e.g., TRPV4, TRPC1, and TRPP2) also exist at the EC membrane and can be activated by mechanical stimuli such as stretch and flow induced-shear stress to regulate endothelial [Ca2+] and cell membrane potential (
To investigate dimensions (e.g., length and diameter) of primary cilia, various techniques have been employed including transmission electron microscopy, scanning electron microscopy (
Due to its convenience, easiness in sample preparation and capability in identifying molecular composition, fluorescent confocal microscopy is the most commonly used technique in determining the ciliary length (
Endothelial primary cilia have been investigated in different species and in different types of ECs located at aortas, arteries, arterioles, veins, and microvessels, as well as under different physiological and pathological conditions. Although their lengths vary from 0.5 to over 15 μm, endothelial primary cilia have a constant diameter of ~0.2 μm (
The length of endothelial primary cilia varies among various types of ECs and at the different locations of the vascular system. As proposed by
The length of cilia can change during injury and inflammation. For example, the length of cilia increases when exposed to pro-inflammatory cytokines (
Cell type | Length (μm) | References |
---|---|---|
Bovine aortic endothelial cells | 0.7–6.1 | |
Mouse embryonic aorta endothelial cells | 2.1–6.2 | |
Porcine coronary artery endothelial cells | 0.5–2.3 | |
Mouse femoral artery endothelial cell | 0.5–0.8 | |
Endothelial-like cell from the bulbus arteriosus | 1.1–3.4 | |
Human umbilical vein endothelial cells | 1.8–11.1 | |
0.5–5.3 |
||
Human microvascular endothelial cell | 1.1–16.5 | |
Human pulmonary microvascular endothelial cells | 1.2–3.5 | |
Mouse embryonic endothelial cells | 1.7–4.1 | |
Zebrafish embryonic endothelial cells | 3.4–3.9 |
Besides structure, molecular composition and dimension, mechanical properties, e.g., stiffness or bending rigidity, are essential for the primary cilium to serve as a blood flow sensor and transducer. The mechanical properties of primary cilia can be determined by combining the bending behavior of the cilium recorded by the image-based techniques and the mathematical models for the force-deflection of cilia (
The bending rigidity of the cilium can also be obtained by using an optical trap with a magnetic bead affixed to the primary cilium (
The mathematical models for the cilium-fluid flow (or other applied forces) are necessary in determining the mechanical properties of the cilia.
So far, almost all the studies investigating the mechanical properties of the cilia that we can find are on the epithelial cells or other types of cells except one on the endothelial cells. As described above, the bending rigidity and spring constants are the parameters charactering the mechanical properties of primary cilia, we thus list in
Cell type | Parameter | Value | References |
---|---|---|---|
mCCD 1296 (d) | Linear spring constant | (4.6 ± 0.62) × 10−12 N/rad | |
mCCD 1296 (d) | Nonlinear spring constant | (−1 ± 0.34) × 10−10 N/rad2 | |
mCCD 1296 (d) | Bending rigidity | (1–2) × 10−23 Nm2 | |
IMCD | Bending rigidity | (1–5) ×10−23 Nm2 | |
MDCK | Bending rigidity | (0.9–1.96) ×10−23 Nm2 | |
EC | Bending rigidity | (0.5–1) × 10−23 Nm2 |
Note: Abbreviations: mCCD 1296 (d): a mouse cell line derived from the cortical collecting duct; IMCD: inner medullary collecting-duct kidney epithelial cells; MDCK: Madin-Darby canine kidney cells; EC: endothelial cells from the dorsa aorta of zebrafish.
Protruding from the middle of the apical surface of an EC into the vessel lumen, the endothelial primary cilium has the best position to sense the blood flow. It has thus been widely recognized as one of the prominent mechano-sensors and mechano-transducers, which could sense and respond to the blood flow and convert the blood flow induced mechanical stimuli into biochemical signaling through its transmembrane proteins and other accessories. In fact, primary cilia are linked to the EC cytoskeleton via the microtubule-organizing center. As a result, the torque exerted on the primary cilium by the blood flow can be transmitted throughout the cell. Meanwhile, there are many other mechano-sensors located on the EC, which can be affected and activated by the alteration of cytoskeletons caused by bending of primary cilia. Although the exact molecular mechanism by which primary cilia act as a blood flow sensor and transducer is still under investigation, the proper mechanical properties (reviewed in the section above) and adequate mechanical strength of the primary cilium is essential for this duty.
It has been found that endothelial primary cilia participate in regulating actin organization, focal adhesion formation, directional migration, vascular development, and cell permeability via flow sensing (
In contrast, high shear stresses (HSS) may cause disassembly of primary cilia both
The generation of primary cilia, or ciliation, is highly dependent on the flow pattern rather than the flow magnitude.
As expected, many molecules should be coordinated for the primary cilia to perform their functions. By flow sensing, endothelial primary cilia can regulate actin organization, focal adhesion formation, directional migration, and cell permeability via heat-shock protein 27 (hsp27) pathway (
In addition to increasing cytosolic [Ca2+] acutely upon the flow, the antenna-like cilium is thought to amplify the cytoskeletal strain and exert a prolonged effect on the expression of shear responsive transcription factors, including Krüppel–like factor 2 (Klf2) and Krüppel–like factor 4 (Klf4) (
Endothelial primary cilia also play a role in maintaining normal morphology of ECs lining vascular walls. Under fluid shear stresses (FSS), the wild-type ciliated ECs retain cobblestone morphology, while the non-ciliated ECs undergo endothelial-mesenchymal transition (EndoMT) and become spindle-shaped (
Besides morphological maintenance of ECs under blood flows, endothelial primary cilia contribute to EC directional migration and vascular stability.
Endothelial primary cilia also mediate Wnt, Sonic Hedgehog (SHH) activity and Notch signaling pathways during EC development and function (
Independent of mechano-sensing and transduction, due to its unique location, dimension, structure and mechanical properties, the primary cilium has recently proposed to redistribute and reduce the WSS under real physiological pulsatile flows, and thus play a protective role for the ECs lining the blood vessel walls.
Dysfunction of primary cilia induces many diseases including cardiovascular diseases. These diseases are collectively named as ciliopathies. Specifically, malfunction of endothelial primary cilia in shear forces (or tangential forces) sensing causes atherosclerosis, hypertension, and aneurysm formation. This section briefly summarizes the role of endothelial primary cilia in these diseases. Extensive reviews can be found in (
Atherosclerosis plaques preferentially occur at bifurcations, branch points and inner surfaces of arched arteries with relatively low and disturbed blood flows, where primary cilia are usually enriched (
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 genes encoding the proteins polycystin-1 or polycystin-2. The patients with ADPKD also show many cardiovascular manifestations such as hypertension, cardiac valveabnormalities, and pericardial effusions (
An aneurysm is a formation of a swelling in an area of a blood vessel that can rupture, leading to bleeding and possibly to death if occurs in vital organs such as brain. Aneurysm formation and rupture are one of the major complications associated with ADPKD because polycystin-1 and polycystin-2 at endothelial primary cilia are required in blood vessels for proper flow sensing and transduction.
The unique location, structure, mechanical properties and molecular compositions, all of them contribute to the function of endothelial primary cilia in maintaining normal cardiovascular system. As an antenna, the primary cilium can sense the changes in external blood flows and transduce them into ECs via various signaling pathways along the ciliary membrane, cilioplasm, axoneme, and at the basal body complex. Even without the signaling function, the endothelial cilium can still behave like a structural component to redistribute and reduce the flow generated wall shear stresses to protect the vessel wall. The disturbance near the vessel wall by the interaction between the cilium and fluid flow also favors the mass transport at the EC surface to bring nutrients from the bulk flow and remove the metabolic wastes. Lack of primary cilia or malfunction of cilia cause many cardiovascular diseases including atherosclerosis, hypertension, and aneurysm formation.
Although the molecular mechanism by which endothelial primary cilia perform their flow sensing and transducing has been partially elucidated, further investigation is highly expected. With the development of super high resolution optical microscopy, more detailed information for the structural (molecular) components of EC primary cilia can be revealed, especially when the ECs are alive, and the forces are known. As the force and stress distribution on a cilium and other structural and mechanical factors are beyond the capability of current experimental approaches due to the nanometer scales in its structure and the pulsatile nature of the real physiological flows, more sophisticated mathematical models and numerical approaches should be developed. By combining the model predictions with the experimental observations, the more detailed molecular mechanism underlying the cilium sensing-transducing can be elucidated under realistic physiological conditions. In addition, the interactions of primary cilia with other endothelial mechano-sensors and transducers also deserve investigation to give a more comprehensive understanding for how the ECs sense and transduce the external stimuli into internal signals for their functions.
The understanding for how the primary cilia function will also help to design proper therapies for cardiovascular diseases due to lack of cilia or malfunction of cilia. For example, targeted gene therapy for PKD1/PKD2 may reverse the ADPKD and related cardiovascular diseases such as hypertension and aneurysm. Pharmacological agents can be invented to either help in generating primary cilia or in informing alternative flow sensors and transducers on ECs in the absence of cilia. Since pulsatile flows at certain levels can help regenerate lost primary cilia, proper exercises can be designed for this purpose.