To explore the changes of wall shear stress(WSS) effect on arterial endothelial cell(EC) apoptosis after reducing arterial blood flow. The reducing flow model was established in 60 rabbits. Endothelial stretched preparations were made at 8 different time intervals from 0 to 30 days. The apoptosis rate of arterial endothelial cells (AEC) was measured with TdT-mediated dUTP-biotin nick end labeling(TUNEL) method. The results showed that the apoptosis rate of AEC was significantly higher from 1 day to 7 days after decreasing WSS than that of control, which peaked on day 3. While with progressively increasing in WSS, the apoptosis rate restored to the level of control from 14 days to 30 days. These suggest that the apoptosis state of AEC might be markedly influenced by the changes of WSS. The persist decreasing of WSS may be the important factor which induces the cell apoptosis.
Animals ; Apoptosis ; Arteries ; cytology ; physiology ; Endothelium, Vascular ; cytology ; physiology ; Male ; Rabbits ; Regional Blood Flow ; Shear Strength

This study was aimed to compare the differences of adhesion properties of endothelial cells (EC) from arteries (AEC), veins (VEC) and capillaries (MVEC) under shear stress condition, and to explore whether they can get the same adhesive ability as graft in similar shear stress conditions. With mended parallel plate flow apparatus and peristalsis pump providing fluid shear stress used, endothelial culture models were established in vitro with the same environmental factors as steady culture. To compare the adhesion among three kinds of endothelial cells under dynamic condition and static condition, the dynamic change of cytoskeletal actin filaments and the effects of different adhesive proteins coated on the adhesion of EC to the glass were studied. The cultured endothelial cells under flow conditions were extended and arranged along the direction of flow. The adhesive ability from high to low under static condition were AEC, MVEC and VEC (VEC compared with AEC or MVEC, P < 0.05), sequentially. The adhesion of endothelial cells from variety sources under dynamic culture condition was significantly increased than that of the static groups. The ratio of cell retention was not significantly different between AEC and MVEC. But VEC was significantly different (P < 0.05) compared with AEC or MVEC. The actin filaments (F-actin) were bundled together and arranged along the direction of flow after fluid culture. Dense peripheral band (DPB) gradually disappeared and distinct stress fibers were formed, which even interconnected to form a whole in the MVEC. The adhesion of AEC, VEC and MVEC under shear stress conditions are more significantly increased than those under the static culture conditions, and the MVEC can achieve the same adhesion as AEC.
Arteries ; cytology ; Capillaries ; cytology ; Cell Adhesion ; Cell Culture Techniques ; methods ; Cells, Cultured ; Cytoskeleton ; physiology ; Endothelial Cells ; cytology ; physiology ; Humans ; Shear Strength ; Stress, Mechanical ; Veins ; cytology

OBJECTIVEReduced arterial elasticity is a hallmark of aging in healthy humans independently of diseases and endothelial-cell injury and dysfunction may be responsible for this fall in arterial elasticity. We hypothesized that circulating endothelial progenitor cells (EPCs) are involved in endothelial repair and that lack of EPCs contributes to impaired arterial elasticity.

METHODSA total of 56 healthy male volunteers were divided into young (n = 26) and elderly (n = 30) groups. Large and small artery elasticity indices were non-invasively assessed by using pulse wave analysis. Flow cytometer was used to count the number of circulating CD34(+) mononuclear cells (MNCs), which were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. EPCs were characterized as adherent cells double positive staining for DiI-acLDL uptake and lectin binding with using fluorescent microscope.

RESULTSC(1) (large artery elasticity index) and C(2) (small artery elasticity index) were significantly reduced in the elderly group compared with those in the young group (11.73 +/- 1.45 vs 16.89 +/- 1.69 ml/mm Hg x 10, P < 0.001; 8.40 +/- 1.45 vs 10.58 +/- 1.18 ml/mm Hg x 100, P < 0.001 respectively). In parallel, the number of circulating EPCs was significantly reduced in the elderly group compared with the young group (0.13 +/- 0.02 vs 0.17 +/- 0.04%, P < 0.05). The number of circulating EPCs correlated with C(1) large and C(2) small artery elasticity indices (r = 0.47, P < 0.01; r = 0.4, P < 0.01). Fluorescent microscope was used to identify EPCs, which were double positive staining for DiI-acLDL uptake and lectin binding.

CONCLUSIONThe present findings suggested that the fall in circulating EPCs with subsequently impaired endothelial-cell repair and function might contribute to reduced arterial elasticity in humans with aging. The decrease in circulating EPCs could serve as a surrogate biologic measure of vascular function and human age.

Adult ; Aged ; Aging ; physiology ; Arteries ; physiology ; Elasticity ; Endothelial Cells ; cytology ; physiology ; Humans ; Male ; Middle Aged ; Stem Cells ; cytology ; physiology

Arterioles are major contributors to the control of systemic blood pressure and local blood flow. In this study, we compared electrophysiological properties of vascular smooth muscle cells (VSMCs) in anterior inferior cerebellar artery (AICA), mesenteric artery (MA) and spiral modiolar artery (SMA) by intracellular microelectrode recording and whole-cell patch clamp recording techniques. Results were shown as below: (1) Intracellular microelectrode recordings were made from VSMCs in AICA, MA and SMA with resting potentials of (-68±1.8) (n=65), (-71±2.4) (n=80) and (-66±2.9) mV (n=58), respectively. There was no significant difference in resting potentials among arterioles. (2) The membrane capacitance and membrane conductance in situ cells were much larger than those in dispersed smooth muscle cells by whole-cell recording techniques, and there was significant difference among arterioles, which were in the order: MA>AICA>SMA. After application of gap junction blocker 2-APB (100 μmol/L), the membrane capacitance and membrane conductance in situ cells were very close with those in single smooth muscle cells. (3) The I/V relation of whole-cell current of dissociated smooth muscle cells (AICA, MA and SMA) showed a prominent outward rectification, and the currents were substantially inhibited by 1 mmol/L 4-AP or 10 mmol/L TEA. When the command voltage was +40 mV, the current densities of VSMCs in AICA, MA and SMA were (26±2.0), (24±1.7) and (18±1.3) pA/pF respectively. SMA showed significant difference in the current density from AICA and MA respectively. These results suggest that the electrophysiological properties of coupling strength of gap junction and current density of smooth muscle cells are different among arterioles in the guinea pig.
Animals ; Arterioles ; cytology ; physiology ; Cerebellum ; blood supply ; Cochlea ; blood supply ; Electrophysiological Phenomena ; Female ; Guinea Pigs ; Male ; Mesenteric Arteries ; cytology ; Muscle, Smooth, Vascular ; cytology ; physiology

Laser scanning confocal microscopy (LSCM) and whole-cell perforated patch-clamp techniques were combined to study simultaneously the changes of intracellular signal molecules and membrane currents. Intracellular calcium transients and spontaneous transient outward currents (STOCs) were recorded simultaneously in freshly isolated mouse cerebral artery smooth muscle cells. The cells loaded with fluo-4/AM were scanned with the confocal line-scan mode. Triggering voltage pulses derived from an EPC-10 patch clamp amplifier triggered the confocal line scan. The results showed that STOCs and intracellular calcium transients could be simultaneously recorded in the same cell. This technique will be useful in studies of diseases caused by impairments of intracellular Ca(2+) signaling and related ionic channel activities, or vice versa.
Animals ; Calcium Signaling ; Cerebral Arteries ; cytology ; Mice ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques

It has been reported that a change in the cellular redox state may be involved in the regulation of vascular tone, but the underlying mechanism is not fully understood. The present study was designed to investigate the cellular effect of sulfhydryl modifying agents in the coronary artery of rabbit using the tension measurement and whole cell clamping method. The application of diamide, a sulfhydryl oxidizing agent, relaxed the endothelium denuded coronary arteries in a dose dependent manner. The fact that this diamide-induced relaxation was significantly attenuated by a pretreatment of 4-AP, and the coronary arteries precontracted with 100 mM K+ instead of histamine, suggests the involvement of 4-AP sensitive K+ channels in the diamide-induced relaxation of coronary arteries. Whole cell patch clamp studies revealed that the 4-AP sensitive IdK was significantly enhanced by the membrane permeant oxidizing agents, diamide and DTDP, and were reversed by subsequent exposure to the reducing agent, DTT. Neither the membrane impermeant oxidizing or reducing agents, GSSG or GSH, had any effect on the activity of IdK, indicating that intracellular sulfhydryl modification is critical for modulating IdK activity. The Diamide failed to significantly alter the voltage dependence of the activation and inactivation parameters, and did not change the inactivation process, suggesting that diamide increases the number of functional channels without altering their gating properties. Since IdK has been believed to play an important role in regulating membrane potential and arterial tone, our results about the effect of sulfhydryl modifying agents on coronary arterial tone and IdK activity should help understand the pathophysiology of the diseases, where oxidative damage has been implicated.
Animal ; Arteries/physiology ; Arteries/drug effects ; Arteries/cytology ; Coronary Vessels/physiology ; Coronary Vessels/drug effects* ; Coronary Vessels/cytology ; Female ; Male ; Oxidants/pharmacology* ; Potassium Channels/physiology ; Rabbits ; Reducing Agents/pharmacology* ; Sulfhydryl Compounds/metabolism*

To explore primarily the reconstruction of biological tissue engineered blood vessel in vitro, the decellularised scaffolds were obtained from swine common carotid arteries by enzyme digestion, and vascular smooth muscle cells (VSMCs) were isolated from canine thoracic aorta and subcultured and purified. The VSMCs were seeded into the inner surface of scaffolds and they were cocultured in vitro for 4 weeks. Histological staining and transmission electron microscopy were used to observe the growth of canine VSMCs on swine decellularized arterial scaffolds. The results showed the seeded VSMCs grew well on the decellularized scaffold throughout the duration of 4 weeks, which suggests that the method in this article is practicable for reconstructing complete biological tissue engineering blood vessel.
Animals ; Arteries ; Blood Vessel Prosthesis ; Cells, Cultured ; Coculture Techniques ; Dogs ; Microscopy, Electron, Transmission ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Swine ; Tissue Engineering ; Tissue Scaffolds

The silicone tube flow chamber system has been used to study the effects of wall shear stress and circumferential stress on the cultured vascular endothelial cells (ECs). In solviong the problem of how to precisely simulate the wall shear stress and circumferential stress to which ECs are subjected under physiological conditions, it is very essential to select not only the appropriate geometrical and mechanical characteristic but also the proper preload and after-load of the silicone tube flow chamber. Firstly, a method to obtain the geometrical and mechanical characteristic of the chamber was given. Secondly, the procedure to simulate the two main mechanical stimuli under the physiological environment was proposed. Finally, the factors controlling the wall shear stress and circumferential stress were summarized.
Arteries ; cytology ; Biomechanical Phenomena ; Blood Flow Velocity ; physiology ; Cell Culture Techniques ; instrumentation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Humans ; Mechanotransduction, Cellular ; physiology ; Shear Strength ; Silicones ; Stress, Mechanical

The changes in potassium currents of vascular smooth muscle cells (VSMCs) isolated from saphenous arteries and the 2nd-6th order branches of the mesenteric arteries of 4-week tail-suspended rats (SUS) were examined using whole cell patch clamp technique. The resting potential (RP) of the VSMCs from SUS group was more negative compared with that of the control group (CON).The whole cell potassium current densities of VSMCs isolated from the saphenous arteries and small mesenteric arteries in SUS group were significantly larger than those of the CON group.The BK(Ca) and K(V) current densities of VSMCs from saphenous arteries and small mesenteric arteries from SUS group were also significantly larger than those from the CON group.It is speculated that the hyperpolarization of VSMCs and decreased calcium influx through voltage-dependent calcium channels might be one of the electrophysiological mechanisms involved in the depressed vasoreactivity of hindquarter arteries induced by simulated weightlessness.
Animals ; Arteries ; cytology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Patch-Clamp Techniques ; Potassium ; metabolism ; Potassium Channels, Calcium-Activated ; physiology ; Rats ; Rats, Sprague-Dawley ; Weightlessness Simulation

Vascular smooth muscle cells (VSMCs) are crucial for studying the mechanism and regulation of vasogenic response, but so far no ideal method has been reported for their isolation. We established a VMSC isolation method based on two-step digestion, with which a large population of single smooth muscle cells could be obtained. After cell isolation, the potassium current, cytosolic calcium concentration, reactivity to caffeine were measured, and the results demonstrated that the cells were highly viable and exhibited reactivity to the vascular constrictor to meet various needs in experimental research.
Animals ; Calcium ; metabolism ; Cell Separation ; methods ; Female ; Male ; Membrane Potentials ; Mesenteric Arteries ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; physiology ; Patch-Clamp Techniques ; Potassium Channels ; physiology ; Rats ; Rats, Wistar