Medicinal plant rosemary relaxes blood vessels by activating vascular smooth muscle KCNQ channels.

The evergreen plant rosemary (Salvia rosmarinus) has been employed medicinally for centuries as a memory aid, analgesic, spasmolytic, vasorelaxant and antihypertensive, with recent preclinical and clinical evidence rationalizing some applications. Voltage-gated potassium (Kv) channels in the KCNQ (Kv7) subfamily are highly influential in the nervous system, muscle and epithelia. KCNQ4 and KCNQ5 regulate vascular smooth muscle excitability and contractility and are implicated as antihypertensive drug targets. Here, we found that rosemary extract potentiates homomeric and heteromeric KCNQ4 and KCNQ5 activity, resulting in membrane hyperpolarization. Two rosemary diterpenes, carnosol and carnosic acid, underlie the effects and, like rosemary, are efficacious KCNQ-dependent vasorelaxants, quantified by myography in rat mesenteric arteries. Sex- and estrous cycle stage-dependence of the vasorelaxation matches sex- and estrous cycle stage-dependent KCNQ expression. The results uncover a molecular mechanism underlying rosemary vasorelaxant effects and identify new chemical spaces for KCNQ-dependent vasorelaxants.

Acute, pro-contractile effects of prorenin on rat mesenteric arteries.

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin-angiotensin-aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca2+ ]i and force were recorded in arteries loaded with Fura-2AM. Spontaneously transient outward currents were recorded via perforated whole-cell patch-clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V-ATPase, caveolin-1, ryanodine receptors, and large conductance Ca2+ -activated K+ channels (BKCa ) in VSMCs. [Ca2+ ]i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1-adrenoreceptor-mediated contraction, associated with an increased number of Ca2+ waves, independent of voltage-gated Ca2+ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BKCa -mediated relaxation. Inhibition of the V-ATPase with 100 nM bafilomycin prevented prorenin-mediated inhibition of BKCa -derived relaxation. Renin (1 nM) had no effect on BKCa -mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BKCa and increasing intracellular Ca2+ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V-ATPase.

Cellular and humoral responses to SARS-CoV-2 vaccination in immunosuppressed patients.

OBJECTIVE: SARS-CoV-2 vaccinations have demonstrated vaccine-immunogenicity in healthy volunteers, however, efficacy in immunosuppressed patients is less well characterised. There is an urgent need to address the impact of immunosuppression on vaccine immunogenicity. METHODS: Serological, T-cell ELISpot, cytokines and immunophenotyping were used to assess vaccine responses (either BNT162b2 mRNA or ChAdOx1 nCoV-19) in double-vaccinated patients receiving immunosuppression for renal transplants or haematological malignancies (n = 13). Immunological responses in immunosuppressed patients (VACC-IS) were compared to immunocompetent vaccinated (VACC-IC, n = 12), unvaccinated (UNVACC, n = 11) and infection-naïve unvaccinated (HC, n = 3) cohorts. RESULTS: No significant different differences in T-cell responses were observed between VACC-IS and VACC-IC (92%) to spike-peptide (S) stimulation. UNVACC had the highest T-cell non-responders (n = 3), whereas VACC-IC and VACC-IS both had one T-cell non-responder. No significant differences in humoral responses were observed between VACC-IC and VACC-IS, with 92% (12/13) of VACC-IS patients demonstrating seropositivity. One VACC-IS failed to seroconvert, however had detectable T-cell responses. All VACC-IC participants were seropositive for anti-spike antibodies. VACC-IS and VACC-IC participants elicited strong Th1 cytokine response with immunodominance towards S-peptide. Differences in T-cell immunophenotyping were seen between VACC-IS and VACC-IC, with lower CD8+ activation and T-effector memory phenotype observed in VACC-IS. CONCLUSION: SARS-CoV-2 vaccines are immunogenic in patients receiving immunosuppressive therapy, with responses comparable to vaccinated immunocompetent participants. Lower humoral responses were seen in patients treated with B-cell depleting therapeutics, but with preserved T-cell responses. We suggest further work to correlate both protective immunity and longevity of these responses in both healthy and immunosuppressed patients.

Feasibility of a mean platelet volume standard: an international council for standardization in hematology (ICSH) inter-laboratory study.

We have evaluated a commercial-fixed porcine platelet preparation (with and without added fixed human red blood cells (RBC)) for the potential standardization of mean platelet volume (MPV) measurements. The standards (Biotechne) were distributed internationally to 19 laboratories including all major hematology instrument manufacturers and academic/pathology laboratories. Overall, the standards demonstrated excellent stability up to 1 month within both MPV values and platelet counts when stored at 4°C. The presence of RBC significantly increased the platelet count and MPV values compared to platelets alone. However, as expected, there were differences in MPV values between different instruments and manufacturers. MPV values were also significantly higher in the whole blood standard compared to the platelet standard in the majority of instruments except with some instruments, where MPV values were significantly higher in the platelet only preparation. To further investigate this phenomenon, two different Platelet MPV preparations (with low and high MPV) in combination with 3 different RBC MCV preparations (with low, normal or high MCVs) were tested to try and further elucidate how RBC populations may impact upon platelet analysis (count, MPV, and PDW) using a single impedance analyzer. Both MPV and MCV values showed good stability over the course of the study for up to 50 days. As expected, the RBC preparation with the lowest MCV had the greatest impact on the MPV. However, this was not observed with an increase in MCV of the RBC or by a larger MPV of the platelet population. To further understand how different gating strategies may also influence results, we investigated the effect of either fixed or floating gate strategies upon MPV raw data from patient samples in a single impedance analyzer. Overall, it was clear that floating and fixed gate strategies also significantly impact upon MPV values. In conclusion, we have demonstrated the potential of an MPV standard with good stability characteristics for calibrating and comparing full blood counters that use different analysis principles, gating and MPV calculations. This may facilitate future instrument calibration and harmonization of results between different technologies.

A small molecule inhibitor of the chloride channel TMEM16A blocks vascular smooth muscle contraction and lowers blood pressure in spontaneously hypertensive rats.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action.

SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels.

OBJECTIVE: The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells. CONCLUSIONS: SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.

High spatial resolution (1.1 µm and 20 nm) FTIR polarization contrast imaging reveals pre-rupture disorder in damaged tendon.

Collagen is a major constituent in many life forms; in mammals, collagen appears as a component of skin, bone, tendon and cartilage, where it performs critical functions. Vibrational spectroscopy methods are excellent for studying the structure and function of collagen-containing tissues, as they provide molecular insight into composition and organization. The latter is particularly important for collagenous materials, given that a key feature is their hierarchical, oriented structure, organized from molecular to macroscopic length scales. Here, we present the first results of high-resolution FTIR polarization contrast imaging, at 1.1 µm and 20 nm scales, on control and mechanically damaged tendon. The spectroscopic data are supported with parallel SEM and correlated AFM imaging. Our goal is to explore the changes induced in tendon after the application of damaging mechanical stress, and the consequences for the healing processes. The results and possibilities for the application of these high-spatial-resolution FTIR techniques in spectral pathology, and eventually in clinical applications, are discussed.

Nanomechanical mapping of hydrated rat tail tendon collagen I fibrils.

Collagen fibrils play an important role in the human body, providing tensile strength to connective tissues. These fibrils are characterized by a banding pattern with a D-period of 67 nm. The proposed origin of the D-period is the internal staggering of tropocollagen molecules within the fibril, leading to gap and overlap regions and a corresponding periodic density fluctuation. Using an atomic force microscope high-resolution modulus maps of collagen fibril segments, up to 80 µm in length, were acquired at indentation speeds around 10(5) nm/s. The maps revealed a periodic modulation corresponding to the D-period as well as previously undocumented micrometer scale fluctuations. Further analysis revealed a 4/5, gap/overlap, ratio in the measured modulus providing further support for the quarter-staggered model of collagen fibril axial structure. The modulus values obtained at indentation speeds around 10(5) nm/s are significantly larger than those previously reported. Probing the effect of indentation speed over four decades reveals two distinct logarithmic regimes of the measured modulus and point to the existence of a characteristic molecular relaxation time around 0.1 ms. Furthermore, collagen fibrils exposed to temperatures between 50 and 62°C and cooled back to room temperature show a sharp decrease in modulus and a sharp increase in fibril diameter. This is also associated with a disappearance of the D-period and the appearance of twisted subfibrils with a pitch in the micrometer range. Based on all these data and a similar behavior observed for cross-linked polymer networks below the glass transition temperature, we propose that collagen I fibrils may be in a glassy state while hydrated.

Hypovitaminosis D, objective oral dryness, and fungal hyphae as three precipitating factors for a subset of secondary burning mouth syndrome.

Objective: Elucidating the concurrence and interdependence of three precipitating factors as contributors of a subset of secondary burning mouth syndrome (BMS), which is defined having detectable precipitating factors. Design: 47 secondary BMS and 15 non-BMS cases were sourced from medical records of an Oral Pathology Specialty Clinic in Canada (2017-2021). Each case had Cytology, Hematology, and Sialometry tests to detail the state of three precipitating factors (the presence of fungal hyphae, hypovitaminosis D, and objective oral dryness). Three factors were compared between secondary BMS and non-BMS groups independently, in pairs, and as a triple-factor by Fisher's exact tests, Contingency Coefficients, and Logistic Regressions. Results: Rates of objective oral dryness (89.36%) and hypovitaminosis D (74.47%) in the secondary BMS group significantly differ from the non-BMS group (p = 0.0013, p = 0.0016). No difference was found in the incidence of fungal hyphae between BMS (91.49%) and non-BMS groups (p = 0.0881). Rates of three precipitating factors in pairs and as a triple-factor within the secondary BMS group significantly differ from the non-BMS group (p-values from 0.0011 to <0.0001). Their significant correlations with secondary BMS are found independently (excluding fungal hyphae), in pairs, and as a triple-factor (C-values from 0.371 to 0.461, p-values from 0.002 to <0.001). The highest C-value belongs to the triple-factor. Objective oral dryness (p = 0.009) and hypovitaminosis D (p = 0.008) are confirmed as significant predictors for secondary BMS. Conclusions: The presence of fungal hyphae contribute to a subset of secondary BMS only when coinciding with objective oral dryness, hypovitaminosis D, or both. This interdependent relationship leads to a hypothesis that hypovitaminosis D, which is commonly called "a low value of vitamin D", and objective oral dryness make an oral environment conducive to insidious Candida invasion, which is an intermediate status of the host-fungal interaction staying between healthy oral mucosa (non-infection) and oral candidiasis (infection).

Cycling matters: Sex hormone regulation of vascular potassium channels.

Sex hormones and the reproductive cycle (estrus in rodents and menstrual in humans) have a known impact on arterial function. In spite of this, sex hormones and the estrus/menstrual cycle are often neglected experimental factors in vascular basic preclinical scientific research. Recent research by our own laboratory indicates that cyclical changes in serum concentrations of sex -hormones across the rat estrus cycle, primary estradiol, have significant consequences for the subcellular trafficking and function of KV. Vascular potassium channels, including KV, are essential components of vascular reactivity. Our study represents a small part of a growing field of literature aimed at determining the role of sex hormones in regulating arterial ion channel function. This review covers key findings describing the current understanding of sex hormone regulation of vascular potassium channels, with a focus on KV channels. Further, we highlight areas of research where the estrus cycle should be considered in future studies to determine the consequences of physiological oscillations in concentrations of sex hormones on vascular potassium channel function.

Marked oestrous cycle-dependent regulation of rat arterial KV 7.4 channels driven by GPER1.

BACKGROUND AND PURPOSE: Kcnq-encoded KV 7 channels (termed KV 7.1-5) regulate vascular smooth muscle cell (VSMC) contractility at rest and as targets of receptor-mediated responses. However, the current data are mostly derived from males. Considering the known effects of sex, the oestrous cycle and sex hormones on vascular reactivity, here we have characterised the molecular and functional properties of KV 7 channels from renal and mesenteric arteries from female Wistar rats separated into di-oestrus and met-oestrus (F-D/M) and pro-oestrus and oestrus (F-P/E). EXPERIMENTAL APPROACH: RT-qPCR, immunocytochemistry, proximity ligation assay and wire myography were performed in renal and mesenteric arteries. Circulating sex hormone concentrations were determined by liquid chromatography-tandem mass spectrometry. Whole-cell electrophysiology was undertaken on cells expressing KV 7.4 channels in association with G-protein-coupled oestrogen receptor 1 (GPER1). KEY RESULTS: The KV 7.2-5 activators S-1 and ML213 and the pan-KV 7 inhibitor linopirdine were more effective in arteries from F-D/M compared with F-P/E animals. In VSMCs isolated from F-P/E rats, exploratory evidence indicates reduced membrane abundance of KV 7.4 but not KV 7.1, KV 7.5 and Kcne4 when compared with cells from F-D/M. Plasma oestradiol was higher in F-P/E compared with F-D/M, and progesterone showed the converse pattern. Oestradiol/GPER1 agonist G-1 diminished KV 7.4 encoded currents and ML213 relaxations and reduced the membrane abundance of KV 7.4 and interaction between KV 7.4 and heat shock protein 90 (HSP90), in arteries from F-D/M but not F-P/E. CONCLUSIONS AND IMPLICATIONS: GPER1 signalling decreased KV 7.4 membrane abundance in conjunction with diminished interaction with HSP90, giving rise to a 'pro-contractile state'.

Sexual dimorphism in prostacyclin-mimetic responses within rat mesenteric arteries: A novel role for KV 7.1 in shaping IP receptor-mediated relaxation.

BACKGROUND AND PURPOSE: Prostacyclin mimetics express potent vasoactive effects via prostanoid receptors that are not unequivocally defined, as to date no study has considered sex as a factor. The aim of this study was to determine the contribution of IP and EP3 prostanoid receptors to prostacyclin mimetic iloprost-mediated responses, whether KV 7.1-5 channels represent downstream targets of selective prostacyclin-IP-receptor agonist MRE-269 and the impact of the oestrus cycle on vascular reactivity. EXPERIMENTAL APPROACH: Within second-order mesenteric arteries from male and female Wistar rats, we determined (1) relative mRNA transcripts for EP1-4 (Ptger1-4 ), IP (Ptgi) and TXA2 (Tbxa) prostanoid receptors via RT-qPCR; (2) the effect of iloprost, MRE-269, isoprenaline and ML277 on precontracted arterial tone in the presence of inhibitors of prostanoid receptors, potassium channels and the molecular interference of KV 7.1 via wire-myograph; (3) oestrus cycle stage via histological changes in cervical cell preparations. KEY RESULTS: Iloprost evoked a biphasic response in male mesenteric arteries, at concentrations ≤100 nmol·L-1 relaxing, then contracting the vessel at concentration ≥300 nmol·L-1 , a process attributed to IP and EP3 receptors respectively. Secondary contraction was absent in the females, which was associated with a reduction in Ptger3. Pharmacological inhibition and molecular interference of KV 7.1 significantly attenuated relaxations produced by the selective IP receptor agonist MRE-269 in male and female Wistar in dioestrus/metoestrus, but not pro-oestrus/oestrus. CONCLUSIONS AND IMPLICATIONS: Stark sexual dimorphisms in iloprost-mediated vasoactive responses are present within mesenteric arteries. KV 7.1 is implicated in IP receptor-mediated vasorelaxation and is impaired by the oestrus cycle.

Multi-pin contact drawing enables production of anisotropic collagen fiber substrates for alignment of fibroblasts and monocytes.

Type I collagen is the most abundant protein in the human body and is known to play important roles in numerous biological processes including tissue morphogenesis and wound healing. As such, it is one of the most frequently used substrates for cell culture, and there have been considerable efforts to develop collagen-based cell culture substrates that mimic the structural organization of collagen as it is found in native tissues, i.e., collagen fibers. However, producing collagen fibers from extracted collagen has been notoriously difficult, with existing methods providing only low throughput production of collagen fibers. In this study, we prepared collagen fibers using a highly efficient, bio-friendly, and cost-effective approach termed contact drawing, which uses an entangled polymer fluid to aid in fiber formation. Contact drawing technology has been demonstrated previously for collagen using highly concentrated dextran solutions with low concentrations of collagen. Here, we show that by replacing dextran with polyethylene oxide (PEO), high collagen content fibers may be readily formed from mixtures of soluble collagen and PEO, a polymer that readily forms fibers by contact drawing at concentrations as low as 0.5%wt. The presence of collagen and the formation of well-ordered collagen structures in the resulting fibers were characterized by attenuated total reflectance Fourier-transform infrared spectromicroscopy, Raman spectromicroscopy, and fluorescence microscopy. Corresponding to well-ordered collagen, the mechanical properties of the PEO-collagen fibers approximated those observed for native collagen fibers. Growth of cells on aligned PEO-collagen fibers attached to a polydimethyl siloxane support was examined for human dermal fibroblast (WS1) and human peripheral leukemia blood monocyte (THP-1) cell lines. WS1 and THP-1 cells readily attached, displayed alignment through migration and spreading, and proliferated on the collagen fiber substrate over the course of several days. We also demonstrated the retrieval of viable cells from the PEO-collagen fiber substrates through enzymatic digestion of the collagen substrate with collagenase IV.

Monitoring Chemistry In Situ with a Smart Stirrer: A Magnetic Stirrer Bar with an Integrated Process Monitoring System.

Inspired by the miniaturization and efficiency of the sensors for telemetry, we have developed a device that provides the functionalities of laboratory magnetic stirring and integrated multisensor monitoring of various chemical reaction parameters. The device, called "Smart Stirrer", when immersed in a solution, can in situ monitor physical properties of the chemical reaction such as the temperature, conductivity, visible spectrum, opaqueness, stirring rate, and viscosity. This data is transmitted real-time over a wireless connection to an external system, such as a PC or smartphone. The flexible open-source software architecture allows effortless programming of the operation parameters of the Smart Stirrer in accordance with the end-user needs. The concept of the Smart Stirrer device with an integrated process monitoring system has been demonstrated in a series of experiments showing its capability for many hours of continuous telemetry with fine accuracy and a high data rate. Such a device can be used in conventional research laboratories, industrial production lines, flow reactors, and others where it can log the state of the process to ensure repeatability and operational consistency.

Material properties of disulfide-crosslinked hyaluronic acid hydrogels influence prostate cancer cell growth and metabolism.

Cells reside in vivo within three dimensional environments in which they interact with extracellular matrices (ECMs) that play an integral role in maintaining tissue homeostasis and preventing tumour growth. Thus, tissue culture approaches that more faithfully reproduce these interactions with the ECM are needed to study cancer development and progression. Many materials exist for modeling tissue environments, and the effects of differing mechanical, physical, and biochemical properties of such materials on cell behaviour are often intricately coupled and difficult to tease apart. Here, an optimized protocol was developed to generate low reaction volume disulfide-crosslinked hyaluronic acid (HA) hydrogels for use in cell culture applications to relate the properties of ECM materials to cell signalling and behaviour. Mechanically, HA hydrogels are comparable to other soft hydrogel materials such as Matrigel and agarose or to tissues lacking type I collagen and other fibrillar ECM components. The diffusion of soluble materials in these hydrogels is affected by unique mass transfer properties. Specifically, HA hydrogel concentration affects the diffusion of anionic particles above 500 kDa, whereas diffusion of smaller particles appears unimpeded by HA content, likely reflecting hydrogel pore size. The HA hydrogels have a strong exclusion effect that limits the movement of proteins into and out of the material once fully formed. Such mass transfer properties have interesting implications for cell culture, as they ultimately affect access to nutrients and the distribution of signalling molecules, affecting nutrient sensing and metabolic activity. The use of disulfide-crosslinked HA hydrogels for the culture of the model prostate cancer cell lines PC3 and LNCaP reveals correlations of protein activation linked to metabolic flux, which parallel and can thus potentially provide insights into cell survival mechanisms in response to starvation that occurs in cancer cell microenvironments.

KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells.

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded kV7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

A new longitudinal variation in the structure of collagen fibrils and its relationship to locations of mechanical damage susceptibility.

The hierarchical architecture of the collagen fibril is well understood, involving non-integer staggering of collagen molecules which results in a 67 nm periodic molecular density variation termed D-banding. Other than this variation, collagen fibrils are considered to be homogeneous at the micro-scale and beyond. Interestingly, serial kink structures have been shown to form at discrete locations along the length of collagen fibrils from some mechanically overloaded tendons. The formation of these kinks at discrete locations along the length of fibrils (discrete plasticity) may indicate pre-existing structural variations at a length scale greater than that of the D-banding. Using a high velocity nanomechanical mapping technique, 25 tendon collagen fibrils, were mechanically and structurally mapped along 10 µm of their length in dehydrated and hydrated states with resolutions of 20 nm and 8 nm respectively. Analysis of the variation in hydrated indentation modulus along individual collagen fibrils revealed a micro-scale structural variation not observed in the hydrated or dehydrated structural maps. The spacing distribution of this variation was similar to that observed for inter-kink distances seen in SEM images of discrete plasticity type damage. We propose that longitudinal variation in collagen fibril structure leads to localized mechanical susceptibility to damage under overload. Furthermore, we suggest that this variation has its origins in heterogeneous crosslink density along the length of collagen fibrils. The presence of pre-existing sites of mechanical vulnerability along the length of collagen fibrils may be important to biological remodeling of tendon, with mechanically-activated sites having distinct protein binding capabilities and enzyme susceptibility.

Chronic, programmed polypeptide delivery from an implanted, multireservoir microchip device.

Implanted drug delivery systems are being increasingly used to realize the therapeutic potential of peptides and proteins. Here we describe the controlled pulsatile release of the polypeptide leuprolide from microchip implants over 6 months in dogs. Each microchip contains an array of discrete reservoirs from which dose delivery can be controlled by telemetry.

MMP-9 selectively cleaves non-D-banded material on collagen fibrils with discrete plasticity damage in mechanically-overloaded tendon.

The mechanical properties of tendon are due to the properties and arrangement of its collagen fibril content. Collagen fibrils are highly-organized supermolecular structures with a periodic banding pattern (D-band) indicative of the geometry of molecular organization. Following mechanical overload of whole tendon, collagen fibrils may plastically deform at discrete sites along their length, forming kinks, and acquiring a fuzzy, non-D-banded, outer layer (shell). Termed discrete plasticity, such non-uniform damage to collagen fibrils suggests localized cellular response at the fibril level during subsequent repair/replacement. Matrix metallo-proteinases (MMPs) are enzymes which act upon the extracellular matrix, facilitating cell mobility and playing important roles in wound healing. A sub-group within this family are the gelatinases, MMP-2 and MMP-9, which selectively cleave denatured collagen molecules. Of these two, MMP-9 is specifically upregulated during the initial stages of tendon repair. This suggests a singular function in damage debridement. Using atomic force microscopy (AFM), a novel fibril-level enzymatic assay was employed to assess enzymatic removal of material by trypsin and MMP-9 from individual fibrils which were: (i) untreated, (ii) partially heat denatured, (iii) or displaying discrete plasticity damaged after repeated mechanical overload. Both enzymes removed material from heat denatured and discrete plasticity-damaged fibrils; however, only MMP-9 demonstrated the selective removal of non-D-banded material, with greater removal from more damaged fibrils. The selectivity of MMP-9, coupled with documented upregulation, suggests a likely mechanism for the in vivo debridement of individual collagen fibrils, following tendon overload injury, and prior to deposition of new collagen.

TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension.

BACKGROUND AND PURPOSE: Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca2+ -activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca2+ -activated chloride channels, TMEM16A, in rat coronary arteries. EXPERIMENTAL APPROACH: We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs). KEY RESULTS: TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16Ainh -A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16Ainh -A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT. CONCLUSIONS AND IMPLICATIONS: In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs.