Vessels Of Rats Research Articles

Ethanol-induced lymphatic endothelial cell permeability via MAP-kinase regulation.

Chronic alcohol alters the immune system enhancing the susceptibility to inflammation, bacterial, and viral infections in alcohol users. We have shown that alcohol causes increased permeability of mesenteric lymphatic vessels in alcohol-fed rats. The mechanisms of alcohol-induced lymphatic leakage are unknown. Endothelial cell monolayer permeability is controlled by junctional proteins complexes called tight junctions (TJ) and adherens junctions (AJ), and each can be regulated by MAPK activation. We hypothesize that ethanol induces lymphatic endothelial cell (LEC) permeability via disruption of LEC TJ through MAPK activation. An in vitro model of rat LECs was used. Ethanol-supplemented medium was added at concentrations of 0, 25, and 50 mM to confluent cells. Resistance-based barrier function, transwell permeability, cell viability, TJ, AJ, and MAPK protein activity, TJ and AJ gene expressions, and the role of p38 MAPK in barrier function regulation were measured. Ethanol increased the permeability of LECs compared to controls that was not associated with decreased cell viability. LECs treated with 50 mM ethanol showed an increase in phosphorylated levels of p38. No significant changes in TJ and AJ gene or protein expressions were observed after ethanol treatment. p38 inhibition prevented ethanol-induced increases in permeability. These findings suggest that p38 may play a role in the regulation of ethanol-induced LEC permeability, but altered permeability may not be associated with decreased TJ or AJ protein expression. Further investigation into junctional protein localization is needed to better understand the effects of ethanol on lymphatic endothelial cell-to-cell contacts and hyperpermeability.

Early Passive Leg Movement Prevents Against the Development of Heart Failure With Preserved Ejection Fraction in Rats.

Exercising was reported by several studies to bring great benefits to heart failure with preserved ejection fraction (HFpEF), which reduced the hospitalization and the mortality of heart failure. However, the underlying mechanism of exercising on HFpEF remains unclear. In the present study, we designed and constructed a device that can perform early passive leg movement (ePLM) in rats and further observed whether treatment of ePLM exerts protective effects on HFpEF of rats. Rats were fed with high salt feed to establish an animal model of pre-clinical diastolic dysfunction (PDD), which would eventually develop into HFpEF, and then treated rats with ePLM. We conducted several experiments to evaluate the conditions of heart and blood vessel. The results show that diastolic functions of heart and blood vessel in rats were significantly improved by treatment of ePLM. We also found that pathological injuries of heart and blood vessel were ameliorated after treatment of ePLM. Moreover, treatment of ePLM decreased the protein levels of Collagen type I, Collagen type III, MMP2, and MMP9 in heart and blood vessel, indicating that cardiac and vascular fibrosis were reduced apparently by treatment of ePLM. Further investigation suggested that treatment of ePLM probably inhibit the activation of TGF-β1/Smad3 signaling pathway as well as promote the activation of Akt/eNOS signaling pathway in high salt diet induced HFpEF. In conclusion, treatment of ePLM alleviated high salt diet induced HFpEF by inhibiting fibrosis via suppressing TGF-β1/Smad3 signaling pathway as well as activating Akt/eNOS signaling pathway, implicating treatment of ePLM as a promising novel non-pharmacological approach for HFpEF.

Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke.

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

Effectiveness of Black Honey as an Antiinflammatory Substance in Rat’s Wound Infected by Staphylococcus aureus

Background: Black honey contains higher flavonoids, phenols, and minerals, which serves as antiinflammatory agents and promote faster wound healing. Purpose: This study aimed to determine the effectiveness of black honey on macrophage cells and blood vessels in rats’ wounds infected with Staphylococcus aureus. Methods: This was an experimental study with a post-test only control group design. This study involved 24 rats divided into 4 groups. The treatment was carried out for 5 days. The rats in the K1 group were wounded (cut) and given aquadest. The rats in the K2 group were wounded (cut), infected with Staphylococcus aureus, and given aquadest. The rats in the P1 group were wounded (cut), infected with Staphylococcus aureus, and treated with 2ml of black honey topically. The rats in the P2 group were wounded (cut), infected with Staphylococcus aureus, and treated with2ml of black honey orally. Observations were made histopathologicaly using hematoxylin-eosin (HE) staining by calculating the number of macrophage and blood vessels, and further using One-Way ANOVA. Result: The number of macrophages and blood vessels between groups was significantly different. The K2 group had the highest mean number of macrophage cells and blood vessels, and the K1 group was the least. The mean number of macrophage cells in the P1 group was more than the K1 group and less than the K2 and P2 groups, both significantly. Conclusion: Black honey was effective in reducing inflammation in wounds infected by Staphylococcus aureus.

Pilot study of minimum occlusive force of vascular clamps on arterial vessels in rats

Our aims were to determine the accuracy of an improved formula for determining the minimum occlusive force (MOF) of a vascular clamp on rats’ abdominal aortas, compare our findings with the calculated theoretical MOF, and provide reference data for clinical research and development of medical instruments that cause minimal damage. We created a vessel closure model and developed a formula for calculating the theoretical MOF of arterial vessels when they are occluded. This formula utilises the blood pressure in the blood vessel, its diameter, and the width of the vascular clamp. We then measured the actual MOF in 24 rat abdominal aortic segments with different diameters and different blood pressures and compared the theoretical and actual MOFs. Analysis of the experimental data showed a probability of 0.315, which means that, under the condition of normal distribution, the difference between the theoretical and actual MOF is not significant at the α = 0.05 level. Thus, the actual measured MOF tended to be consistent with the theoretical MOF calculated by the formula we developed. The improved formula will provide a reference for clinical research and development of medical instruments that cause minimal injury, thus contributing to the development of microsurgery.

Training model for the intraluminal continuous suturing technique for microvascular anastomosis

Microvascular anastomosis is a critical procedure in cerebral bypass surgeries. In some rare cases, the extraluminal interrupted technique is not optimal because the vessels are immobile and cannot be rotated, and anastomosis can be performed effectively through the intraluminal continuous suturing technique. The authors reported the application of the intraluminal continuous suturing technique in microanastomosis training with silicone tube, rat’s common iliac arteries and abdominal aorta. A silicone tube with a diameter of 1.5 mm was used to practice microanastomosis in intraluminal continuous suturing technique. Then the technique was applied in side-to-side, end-to-side anastomoses of common iliac arteries and the end-to-end abdominal aorta anastomoses of rat. The suturing time and patency rates were compared with an alternative intraluminal continuous suturing technique and one-way-up interrupted suturing technique in silicone tube and rat vessel anastomoses. The intraluminal continuous suturing technique could be gained through practicing with silicone tube, and the technique has also been demonstrated effective in side-to-side, end-to-side anastomoses of common iliac arteries of rat and the abdominal aorta end-to-end anastomoses. In all the animal experimental groups with different suturing techniques, there was no difference between the patency rates, all the immediate patency rate was 100%. There was no significant suturing time difference between the two intraluminal continuous suturing techniques, but the two intraluminal continuous suturing techniques were faster than the interrupted technique. The intraluminal continuous suturing technique described in the study could be used as an efficient method for side-to-side, end-to-side and end-to-end anastomosis, especially under the situation the posterior wall of the anastomosis could not be rotated. Proficiency of the technique could be achieved through practicing in laboratory with silicone tube and live animals.

Perivascular macrophages produce type I collagen around cerebral small vessels under prolonged hypertension in rats.

Hypertension leads to structural remodeling of cerebral blood vessels, which has been implicated in the pathophysiology of cerebrovascular diseases. The remodeling and progression of arteriolosclerosis under hypertension involve fibrosis along with the production of type I collagen around cerebral arterioles. However, the source and regulatory mechanisms of this collagen production remain elusive. In this study, we examined if perivascular macrophages (PVMs) are involved in collagen production around cerebral small vessels in hypertensive SHRSP/Izm rats. Immunoreactivity for type I collagen around cerebral small vessels in 12-week-old hypertensive rats tended to higher than those in 4-week-old hypertensive and 12-week-old control rats. In ultrastructural analyses using transmission electron microscopy, the substantial deposition of collagen fibers could be observed in the intercellular spaces around PVMs near the arterioles of rats with prolonged hypertension. In situ hybridization analyses revealed that cells positive for mRNA of Col1a1, which comprises type I collagen, were observed near cerebral small vessels. The Col1a1-positive cells around cerebral small vessels were colocalized with immunoreactivity for CD206, a marker for PVMs, but not with those for glial fibrillary acidic protein or desmin, markers for other perivascular cells such as astrocytes and vascular smooth muscle cells. These results demonstrated that enhanced production of type I collagen is observed around cerebral small vessels in rats with prolonged hypertension and Col1a1 is expressed by PVMs, and support the concept that PVMs are involved in collagen production and vascular fibrosis under hypertensive conditions.

Structured evaluation of a comprehensive microsurgical training program

OBJECTIVES:This study proposed a structured microsurgical training program and evaluated it with the assistance of a large sample of surgeons.METHODS:The practical course comprised 16 sessions of approximately 4 hours each. This included two sessions for suturing rubber gloves and two sessions for suturing arteries, veins, and nerves in chicken thighs. The other sessions were performed on the femoral vessels of rats: 5 sessions for end-to-end arterial anastomosis, 5 for end-to-end venous anastomosis, 1 for arterial grafting, and 1 for end-to-side anastomosis. We conducted a structured assessment of the microsurgical skills in each training session.RESULTS:In this study, 89 surgeons were evaluated. The mean scores for the different procedures were as follows: glove suturing, 33.3±0.59; chicken nerve end-to-end anastomosis, 40.3±0.49; chicken artery suturing, 40.9±0.36; chicken vein suturing, 42.3±0.36; graft interposition, 44.8±0.7; and end-to-side anastomosis, 43.7±0.63 (p<0.05 for all). The chicken thigh suturing scores were significantly higher than the rubber gloves suturing scores (p<0.01). There were no differences between scores of the rat artery and chicken thigh suturing procedures (p=0.24). The rat venous anastomosis scores were higher than the rat arterial anastomosis scores (p=0.02), as were graft interposition scores when compared with end-to-end venous anastomosis scores. The end-to-side anastomosis scores did not differ significantly from the grafting scores (p=0.85). The most common errors were inadequate knotting technique and suture rupture due to inadequate technique (both n=88 [98.9%]).CONCLUSION:We propose a 16-step, progressive microsurgical training program to learn the basic microsurgical techniques comprehensively and reliably. The program was evaluated in a large sample of trainees, and it demonstrated the adequacy of the training sequence and results.

The Functional Availability of Arterial Kv7 Channels Is Suppressed Considerably by Large-Conductance Calcium-Activated Potassium Channels in 2- to 3-Month Old but Not in 10- to 15-Day Old Rats

BackgroundVoltage-gated potassium (Kv) channels, especially Kv7 channels, are major potassium channels identified in vascular smooth muscle cells with a great, albeit differential functional impact in various vessels. Vascular smooth muscle Kv7 channels always coexist with other K channels, in particular with BK channels. BK channels differ in the extent to which they influence vascular contractility. Whether this difference also causes the variability in the functional impact of Kv7 channels is unknown. Therefore, this study addressed the hypothesis that the functional impact of Kv7 channels depends on BK channels.Experimental ApproachExperiments were performed on young and adult rat gracilis and saphenous arteries using real-time PCR as well as pressure and wire myography.Key ResultsSeveral subfamily members of Kv7 (KCNQ) and BK channels were expressed in saphenous and gracilis arteries: the highest expression was observed for BKα, BKβ1 and KCNQ4. Arterial contractility was assessed with methoxamine-induced contractions and pressure-induced myogenic responses. In vessels of adult rats, inhibition of Kv7 channels or BK channels by XE991 or IBTX, respectively enhanced arterial contractility to a similar degree, whereas activation of Kv7 channels or BK channels by retigabine or NS19504, respectively reduced arterial contractility to a similar degree. Further, IBTX increased both the contractile effect of XE991 and the anticontractile effect of retigabine, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991. In vessels of young rats, inhibition of Kv7 channels by XE991 enhanced arterial contractility much stronger than inhibition of BK channels by IBTX, whereas activation of Kv7 by retigabine reduced arterial contractility to a greater extent than activation of BK channels by NS19504. Further, IBTX increased the anticontractile effect of retigabine but not the contractile effect of XE991, whereas NS19504 reduced the effect of retigabine and impaired the effect of XE991.ConclusionKv7 and BK channels are expressed in young and adult rat arteries and function as negative feedback modulators in the regulation of contractility of these arteries. Importantly, BK channels govern the extent of functional impact of Kv7 channels. This effect depends on the relationship between the functional activities of BK and Kv7 channels.

Coronary Collateral Microcirculation Reserve Becomes Vestigial with Aging

Introduction: Our previous study indicated that coronary collateral microcirculation reserve (CCMR), native collaterals, transports blood flow to an ischemic area to reduce ischemic tissue injury. This study aimed to observe the changes of CCMR in the hearts of different month-old rats. Methods: We selected 2-, 8-, 16-, and 24-month-old rats as the research objects to monitor the changes of CCMR in rats with aging. After acute myocardial infarction, lectin-FITC was injected into the femoral vein vessels of rats to mark CCMR vessels in the ischemic area. Results: Results of the lectin-FITC perfusion experiment indicated that the number and collagen IV coverage of CCMR vessels declined with aging. Moreover, data suggested a correlation between endothelial nitric oxide synthase and a decline in the number of CCMR vessels. Conclusion: Aging causes CCMR decline in rats.

Opioidergic regulation of contractile activity of lymphatic vessels

The aim is to study the effect of endogenous opioid peptides on the contractile activity of lymphatic vessels in the white rat. Materials and methods . Under isometric conditions, the dynamics of spontaneous contractile activity of isolated annular segments of rat lymphatic vessels under the action of endogenous opioid peptides (beta-endorphin and endomorphin-1) was record; possible mechanisms of opiates action were studied. Results . The range of β-endorphin level 1×10 –11 –1×10 –8 M resulted in a decrease of the minute productivity of the lymphatic vessels, by 22%, mainly due to a decreasing of single contractions frequency. In the presence of blockers of voltage-gated and ATP-sensitive potassium channels and blockers of eNOS, the inhibitory effect was not manifest. Endomorphin-1, used in the concentration range of 1×10 –10 –1×10 –8 M, stimulated the contractile activity of the lymphatic vessels by an average of 15 %. The stimulatory effect was not manifest when using endomorphin-1 in the presence of a selective NK1 receptor antagonist. Conclusions . The inhibitory effect of β-endorphin is associated with the activation of voltage-dependent and ATP-sensitive potassium channels, endothelium-(NO)-dependent mechanisms. The action of endomorphin-1, stimulating lymphangionic motility, can be carry out through interaction with NK receptors.

Rapidly photocurable silk fibroin sealant for clinical applications

Sealants are useful as agents that can prevent the leakage of gas or nonclotting fluids from damaged tissues and of blood from the vascular system following injury or repair. Various formulations for sealants have been developed and applied clinically, but problems still remain in terms of biocompatibility issues, long crosslinking times and low adhesive properties. Herein, to address these issues, we report a methacrylated silk fibroin sealant (Sil-MAS) with rapidly crosslinkable, highly adhesive and biocompatible properties and demonstrate its versatility as a medical glue. The excellent physical properties of Sil-MAS are revealed via in vitro mechanical tests and ex vivo aorta pressure tests. In addition, in in vivo biological tests on the skin, liver, and blood vessels of rats, Sil-MAS showed a superb hemostatic and adhesive ability, with high biocompatibility. Specifically, Sil-MAS strongly contributed to faster wound healing than commercially available materials. Furthermore, we showed a successful proof of concept that Sil-MAS could serve as an ideal photocuring laparoscopic medical glue in a laceration rabbit model of liver and stomach serosa using a homemade endoscopic device. These findings on the applicability of rapidly photocurable silk fibroin indicate that Sil-MAS is a suitable material to supplant existing sealants, adhesives, or hemostatic agents.

The effect of spleen tyrosine kinase inhibitor R406 on diabetic retinopathy in experimental diabetic rats.

To investigate the effect of spleen tyrosine kinase (Syk) inhibitor R406 on diabetic retinopathy (DR) in diabetic mellitus (DM) rats. Rats were randomized into Normal, DM, DM + 5mg/kg R406 and DM + 10mg/kg R406 groups. DM rats were established via injection of streptozotocin (STZ). One week after model establishment, rats in treatment groups received 5mg/kg or 10mg/kg R406 by gavage administration for 12weeks consecutively, followed by the detection with hematoxylin-eosin (HE) staining, Evans blue angiography, retinal trypsin digestion assay, Western blotting, immunohistochemistry, TUNEL assay, immunofluorescence assay and quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). The retina of DM rats presented different degree of edema, disordered and loose structure, swollen cells with enlarged intercellular space, and dilated and congested capillaries. Besides, the retinal vessels of DM rats showed high fluorescence leakage. However, R406 alleviated the above-mentioned conditions, which was much better with high concentration of R406 (10mg/kg). R406 also reversed the down-regulations of occludin, claudin-5, ZO-1 and the up-regulation of and VEGF in retinal tissues of DM rats; inhibited retinal cell apoptosis; strengthened retinal cell proliferation; and reduced expressions of IL-1β, IL-6, TNF-α and nuclear p65 NF-κB in retinal tissues. The improvement in all these indexes was much more significant in rats of DM + 10mg/kg R406 group than in rats of DM + 5mg/kg R406 group. Syk inhibitor R406 could attenuate retinal inflammation in DR rats via the repression of NF-κB activation.

Effect of bone marrow mesenchymal stem cells on perforator skin flap survival area in rats.

Trans-territory perforator flaps are commonly used to reconstruct large defects of the soft tissues. The distal portion of the flap often becomes necrotic, however, as a result of the jeopardised vasculature of choke zone II. The trophic and vascular regenerative properties of bone marrow mesenchymal stem cells (BMSC) seemed to be a promising approach to prevent flaps becoming ischaemic. The purpose of our study is to evaluate the effects of BMSC on the survival of the three-territory perforator flap. The flap model was created based on the deep circumflex iliac vessel in rats. Eighteen rats were distributed, at random, into three groups. Immediately after the flaps were placed, groups were respectively given a single panniculus carnosus injection at choke zone II of either 1×105 (BMSCslow), 1×106 (BMSCshig) BMSC, or phosphate-buffered saline (PBS). On postoperative day seven, we assessed the gross view of the flap and survival. We also evaluated microvessels by histological examination and angiogenesis-related gene expression by quantitative real-time polymerase chain reaction. After high dosage of BMSC, the flap survival rate, diameter and density of microvessels, vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) levels were significantly higher in the BMSC treatment group than the control group. We therefore confirmed the positive effects of BMSC on the survival of multi-territory perforator flaps.

Potential Protective Effects of the Water-Soluble Chinese Propolis on Hypertension Induced by High-Salt Intake.

High‐salt (HS) intake is closely associated with the ignition and progression of hypertension. The mechanisms might be involved in endothelial dysfunction, nitric oxide deficiency, oxidative stress, and proinflammatory cytokines. Propolis is widely used as a natural antioxidant and is a well‐known functional food for its biological activities, which includes anti‐inflammation, antimicrobial, and liver detoxification. In this study, we successfully replicated a HS diet‐induced hypertensive rat model. We found that in the long‐term HS diet group, the myocardial function of the rats was altered and led to a significant decrease (around 49%) in heart function. However, doses of Chinese water‐soluble propolis (WSP) were found directly proportional (11%, 60%, 91%, respectively) to the myocardial function improvement in hypertensive rats. The results from the blood circulation test and hematoxylin‐eosin stains showed that propolis had protective effects on myocardial functions and blood vessels in hypertensive rats. Also, based on the results of western blot and polymerase chain reaction, WSP effectively regulated Nox2 and Nox4 levels and was responsible for a decrease in reactive oxygen species synthesis. Our findings demonstrate that Chinese WSP has a significant effect on the blood pressure of hypertensive rats and their cardiovascular functions that improved significantly. The improvement in the cardiovascular functions might be related to the process of anti‐oxidation, anti‐inflammation, and the improvements of the endothelial function in hypertensive rats.

Sex‐Specific Protein Expression Differences in Peri‐Lymphatic Adipose of Obese and Lean Zucker Rats.

Lymphatic abnormalities have been reported to accompany obesity, however little is understood about the mechanisms that lead to these changes. To gain novel insight, we performed a proteomic study of the adipose tissue surrounding mesenteric lymphatic vessels in obese and lean Zucker rats of both sexes in combination of a study of passive wall mechanics of isolated mesenteric collecting lymphatic vessels. At 12‐weeks of age, body weights were recorded and the rats were euthanized for collection of mesetneric lymphatic vessels for study plus blood via cardiac puncture and peri‐lymphatic adipose tissue (PLAT) from the mesentery for proteomic analysis. Mesenteric collecting lymphatic vessels were mounted on a pressure myograph and wall thickness and diameter were measured across a physiological pressure range (1–5 cm water). Serum and protein lysate from the adipose were were then analyzed through the RayBiotech Rat Cytokine Array Q67 testing service. The cohort for study included ten obese and ten lean Zucker rats, with equal numbers of males and females in each group. The mean body weights of this cohort were: lean males 275.8 ± 17.9 g; lean females 190.8 ± 6.3 g; obese males 458.4 ± 11.8 g; and obese females 440.0 ± 6.2 g. (P&lt;0.0001). Across both sexes, the lymphatic vessels from obese rats had a significantly greater wall thickness compared to vessels from lean rats. In the proteomic study of PLAT, 5 targets were significantly elevated in obese rats of both sexes (CCL2, galectin‐1, galectin‐3, neuropilin‐1, and PPBP). Additionally, there were 11 targets were elevated in obese males, but not females (b‐NGF, CINC‐3, fractalkine, IL‐6, IL‐13, PDGF‐AA, prolactin receptor, RAGE, TIMP‐1, VEGF‐A, FGF‐BP), and 24 targets that were elevated in obese females, but not males (4‐1BB, adiponectin, B7‐1, CTACK, EphA5, Flt‐3L, Gas‐1, GFR alpha‐1, IL‐1ra, IL‐7, IL‐17F, IL‐22, JAM‐A, neuropilin‐2, NOPE, Notch‐1, Notch‐2, P‐cadherin, prolactin, RANTES, SCF, TIMP‐2, and TWEAK receptor). In serum from obese males, there were 4 targets that were increased (JAM‐A, GM‐CSF, ICAM‐1, neuropilin‐2) and 2 targets decreased (GP130, Ppbp) compared to lean males. In serum from obese females, 1 target was significantly increased (CCL2) and 4 targets were decreased (decorin, fractalkine, NOPE, prolactin) compared to lean females. There were also four targets that were different across the sexes regardless of lean/obese status (FLT‐3L, Gas‐1, GFR‐alpha‐1, TIMP‐1). Analysis of the targets elevated in the PLAT in both males and females with the STRING database revealed that the elevated proteins belong to clusters pertaining to VEGF signaling, positive regulation of cell adhesion, regulation of angiogenesis, and response to stress. Collectively, the results suggest that in addition to some common signaling proteins that are elevated in the PLAT of obese Zucker rats compared to lean counterparts, there are also several differential signals across the sexes. Future studies that may reveal differences in lymphatic function or dysfunction across the sexes are warranted.Support or Funding InformationSupported by NIH R01GM120774 and L40HL097863.

Prenatal caffeine exposure induces down-regulation of the protein kinase A/ryanodine receptor/large-conductance Ca2+-activated K+ pathway in the cerebral arteries of old offspring rats

The current study investigated the long-term effects of prenatal caffeine (Caf) exposure on cerebral vessels of old offspring rats. Pregnant rats were treated with Caf (20 mg/kg, twice daily) or 0.9% normal saline during gestational days 3.5-19.5, and offspring were tested at 24 months old. Vascular functions of middle cerebral arteries and ion channel activities in smooth muscle cells were examined using myograph system and patch-clamp. Prenatal Caf exposure decreased isoprenaline (β-adrenergic agonist)-induced dilatation of the middle cerebral artery in the offspring. Treatment with protein kinase A (PKA) inhibitor reduced isoprenaline-mediated vasodilatation to a greater extent in the control. Forskolin-mediated vasodilatation and membrane hyperpolarization were reduced in the Caf group. Large-conductance Ca-activated K (BKCa) channel inhibitor iberiotoxin significantly attenuated forskolin-induced vasodilatation and reduced depolarization in the control, not in the Caf group. The PKA agonist-activated cell-attached single BKCa currents to a greater extent in the control. The mRNA and protein expression levels of PKA-Cα were decreased. The sensitivity of ryanodine receptors to the PKA agonist was blunted in the Caf group, whereas the mRNA expression of ryanodine receptor 2 subunit was reduced. Voltage/Ca sensitivity of BKCa was decreased accompanied by reduced mRNA and protein expression of BKCa-β1 subunits in the Caf group. PKA agonist-stimulated inside-out BKCa currents were weaker in the Caf group. Prenatal exposure to Caf-affected isoprenaline/forskolin-mediated vascular functions in aged cerebral arteries, related to dysfunction of the PKA/ryanodine receptors/BKCa signaling pathway.

Age‐Associated Changes in Cerebrovascular Function and BK Channel Function are Sex‐Specific

Large conductance potassium (BK) channels are known to regulate the myogenic reactivity, yet, it is not clear if aging alters the myogenic reactivity via the BK channel in males and females. Thus we hypothesize that age‐associated changes in BK channel subunits modulate the myogenic reactivity in a sex‐specific manner. We used vascular reactivity, patch‐clamp, and biochemical methods to measure the myogenic reactivity, BK channel function, and expression, respectively, in cerebral vessels of adult and aged male and female Sprague Dawley rats. The results suggest that aging and ovariectomy (OVX) exaggerated the myogenic reactivity of middle cerebral arteries (MCA) in females but attenuated it in males. Aging induced outward eutrophic remodeling in females but inward hypertrophic remodeling in males. Aging decreased total, Kv, BK channel currents, and spontaneous transient outward currents (STOC) in vascular smooth muscle cells isolated from females, but not in males. Aging increased BKα subunit mRNA and protein content both in males and females. However, aging decreased BKβ1 subunit protein and mRNA levels in females only. In males, BKβ1 mRNA is increased, but protein is decreased. Iberiotoxin‐induced MCA constriction is lower in aged females but higher in aged males. OVX decreased, but chronic supplementation of estradiol (OVX+17βE rats) restored BK channel expression and function. BKβ1 mRNA is decreased in cerebral vessels of OVX rats that is consistent with decreased protein content. However, supplementation with 17βE did not increase mRNA, but protein is not decreased suggesting that post‐translational mechanisms contribute to the preservation of the protein. Overall the results suggest that aging and OVX‐associated downregulation of the BKβ1 expression and function in females exaggerates the myogenic reactivity of MCA. However, age‐associated increase in BK channel function in males attenuates the myogenic reactivity of MCA.Support or Funding InformationThis work was supported by, in part, by a grant from the Intramural Research Support Program from UMMC and AHA Scientist Development Grant 13SDG14000006 (M. R. Pabbidi).

Age-Related Changes in the Role of Calcium-Activated Potassium Channels in Acetylcholine Mediated Dilatation of Pial Arterial Vessels in Rats

Age-related changes in the contribution of endothelial hyperpolarization evoked by activation of intermediate-conductance Ca2+-activated K+ (IKca) channels and NO to acetylcholine-mediated dilatation of pial arterial vessels in rats were studied. Using intravital microphotography (×470), a comparative evaluation of pial arterial responses to acetylcholine chloride (ACh, 10–7 M, 5 min) with and without blockade of IKCa channels (clotrimazole, 10–5 M) and NO (L-NAME, 10–3 M) was carried out in Wistar rats aged 4 and 26 months. The contribution of endothelial hyperpolarization and NO to vascular dilatation was evaluated by changes in the number of dilated arteries and the degree of their response to ACh after application of the blockers. For the latter purpose, the erythrocyte flow width was measured in three individual groups of arteries: small- (< 20 µm), medium- (20–40 µm) and large- (> 40 µm) caliber. The obtained data indicate an age-related decrease in the contribution of endothelial hyperpolarization evoked by activation of IKca channels to ACh-induced dilatation of small-caliber pial arteries in rats. At the same time, the role of this mechanism in medium-caliber arteries increases. Apparently, changes in the vasodilatation mechanisms that occur during aging are due to an increasing role of NO-mediated regulation of endothelium-dependent dilatation responses.

Molecular and Cellular Mechanisms that Induce Arterial Calcification by Indoxyl Sulfate and P-Cresyl Sulfate.

The protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are considered to be harmful vascular toxins. Arterial media calcification, or the deposition of calcium phosphate crystals in the arteries, contributes significantly to cardiovascular complications, including left ventricular hypertrophy, hypertension, and impaired coronary perfusion in the elderly and patients with chronic kidney disease (CKD) and diabetes. Recently, we reported that both IS and PCS trigger moderate to severe calcification in the aorta and peripheral vessels of CKD rats. This review describes the molecular and cellular mechanisms by which these uremic toxins induce arterial media calcification. A complex interplay between inflammation, coagulation, and lipid metabolism pathways, influenced by epigenetic factors, is crucial in IS/PCS-induced arterial media calcification. High levels of glucose are linked to these events, suggesting that a good balance between glucose and lipid levels might be important. On the cellular level, effects on endothelial cells, which act as the primary sensors of circulating pathological triggers, might be as important as those on vascular smooth muscle cells. Endothelial dysfunction, provoked by IS and PCS triggered oxidative stress, may be considered a key event in the onset and development of arterial media calcification. In this review a number of important outstanding questions such as the role of miRNA’s, phenotypic switching of both endothelial and vascular smooth muscle cells and new types of programmed cell death in arterial media calcification related to protein-bound uremic toxins are put forward and discussed.