David S. Weber

Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

Objective—We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. Methods and Results—Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the “wound,” and platelet-derived growth factor (PDGF)–induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. Conclusion—These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

RAPAMYCIN INHIBITS VEGF-INDUCED MICROVASCULAR HYPERPERMEABILITY IN VIVO

Microcirculation (2010) 17, 1–9. doi: 10.1111/j.1549‐8719.2009.00012.x

A Novel Mechanism of Vascular Smooth Muscle Cell Regulation by Notch: Platelet-Derived Growth Factor Receptor-β Expression?

See related article, pages 1483–1491 The Notch family of receptors, Notch1 to -4, are heterodimer transmembrane proteins, consisting of an extracellular domain and a noncovalently linked intracellular domain (ICD). Upon interaction with the DSL family of proteins (Jagged, Delta-like) on neighboring cells, Notch undergoes proteolytic cleavage, which frees the ICD from the plasma membrane. This results in translocation of the ICD into the nucleus, where it forms a complex with the CSL family of transcriptional repressors (CBF1/RBP-Jk), removing the repression and allowing for target gene (Hes, Hey) transcription.1,2 Tissue distribution of the Notch proteins varies widely. Notch1 and -4 are predominantly endothelial, prominent in both arteries and veins, and present in all stages of development (embryonic to adult); the expression of Notch2 is typically confined to pulmonary endothelium, but Notch3 is primarily expressed in adult arterial vascular smooth muscle cells (VSMCs) in large conduit, pulmonary, and systemic resistance arteries.3 This specific pattern of temporal and spatial distribution correlate to diverse functions of the Notch family in vascular development and physiology in vertebrates reported to date.4 In the cardiovascular system, Notch signaling plays a role in several aspects of vascular development, including vasculogenesis, angiogenesis, differentiation, vascular remodeling, and VSMC maturation. Notch1 and -4 signaling appears critical in vasculogenesis and angiogenesis during early development, when it interacts with vascular endothelial growth factor signaling to specify artery–vein differentiation of endothelial cells (ECs). Transgenic mice deficient for Notch1 fail to undergo embryonic angiogenic remodeling, and vascular development is arrested at the primitive undifferentiated plexus, resulting in an embryonic lethal phenotype. Notch2-null mutations likewise result in embryonic lethality characterized by multiple large and small vessel aneurysms. Notch4 deletion results in no obvious phenotypic alteration. Notch3−/− mice are viable; however, they fail to develop proper arterial VSMC phenotype, resulting in vein-like …

Endothelial SK3 channel-associated Ca2+ microdomains modulate blood pressure

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3(-/-) endothelial cells. Moreover, Ca(2+) imaging studies in SK3(-/-) endothelium showed increased Ca(2+) transients with reduced amplitude and duration under basal conditions. Importantly, SK3(-/-) endothelium lacked a distinct type of Ca(2+) dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3(-/-) mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca(2+) dynamics, vascular tone, and blood pressure.

G-Protein βγ Subunit Dimers Modulate Kidney Repair after Ischemia-Reperfusion Injury in Rats

Heterotrimeric G-proteins play a crucial role in the control of renal epithelial cell function during homeostasis and in response to injury. In this report, G-protein βγ subunit (Gβγ) dimer activity was evaluated during the process of tubular repair after renal ischemia-reperfusion injury (IRI) in male Sprague Dawley rats. Rats were treated with a small molecule inhibitor of Gβγ activity, gallein (30 or 100 mg/kg), 1 hour after reperfusion and every 24 hours for 3 additional days. After IRI, renal dysfunction was prolonged after the high-dose gallein treatment in comparison with vehicle treatment during the 7-day recovery period. Renal tubular repair in the outer medulla 7 days after IRI was significantly (P < 0.001) attenuated after treatment with high-dose gallein (100 mg/kg) in comparison with low-dose gallein (30 mg/kg), or the vehicle and fluorescein control groups. Gallein treatment significantly reduced (P < 0.05) the number of proliferating cell nuclear antigen–positive tubular epithelial cells at 24 hours after the ischemia-reperfusion phase in vivo. In vitro application of gallein on normal rat kidney (NRK-52E) proximal tubule cells significantly reduced (P < 0.05) S-phase cell cycle entry compared with vehicle-treated cells as determined by 5′-bromo-2′-deoxyuridine incorporation. Taken together, these data suggest that Gβγ signaling contributes to the maintenance and repair of renal tubular epithelium and may be a novel therapeutic target for the development of drugs to treat acute kidney injury.

Changes in Vascular Reactivity and Endothelial Ca2+ Dynamics with Chronic Low Flow

Disruption of blood flow promotes endothelial dysfunction and predisposes vessels to remodeling and atherosclerosis. Recent findings suggest that spatial and temporal tuning of local Ca2+ signals along the endothelium is vital to vascular function. In this study, we examined whether chronic flow disruption causes alteration of dynamic endothelial Ca2+ signal patterning associated with changes in vascular structure and function. For these studies, we performed surgical PL of the left carotid arteries of mice to establish chronic low flow for 2 weeks; right carotid arteries remained open and served as controls (C). Histological sections showed substantial remodeling of PL compared to C arteries, including formation of neointima. Isometric force measurements revealed increased PE‐induced contractions and decreased KCl‐induced contractions in PL vs C arteries. Endothelium‐dependent vasorelaxation in response to ACh; 10−8 to 10−5 mol/L) was significantly impaired in PL vs C vessels. Evaluation of endothelial Ca2+ using confocal imaging and custom analysis exposed distinct impairment of Ca2+ dynamics in PL arteries, characterized by reduction in active sites and truncation of events, corresponding to attenuated vasorelaxation. Our findings suggest that endothelial dysfunction in developing vascular disease may be characterized by distinct shifts in the spatial and temporal patterns of localized Ca2+ signals.

Comparison of antihyperglycemic effects of creatine and glibenclamide in type II diabetic patients

ZusammenfassungDiese Studie vergleicht die Wirkung von Glibenklamid und Kreatin bei Typ-II-Diabetikern. Die doppelblinde Crossover-Studie mit neu diagnostizierten Typ-II-Diabetikern dauerte 14 Tage. Die Patienten erhielten Kreatin (3 g) oder Glibenklamid (3,5 mg) durch fünf Tage; nach zwei Tagen ohne Therapie bekamen die Patienten die umgekehrte Therapie. Im Rahmen der Studie wurde Glukose, Insulin, C-Peptid und Kreatin im Blut gemessen. Kreatin und Glibenklamid verminderte Glukosenkonzentrationen gegenüber den Basalwerten [-15, 60, 90, 120, 180, and 240 min; mol/l]: 12,6±0,83 vs. 12,2±0,56 vs. 12,1±0,57, 15,3±0,63 vs. 13,5±0,70avs. 13,0±0,57a, 14,8±0,57 vs. 13,8±0,59avs. 13,4± 0,46a, 14,6±0,61 vs. 12,3±0,49bvs. 12,4±0,61a, 12,8±0,76 vs. 10,0±0,40cvs. 10,3±0,41c, and 11,4±0,67 vs. 8,3±0,40cvs. 8,5±0,36c; (ap<0,05; bp<0,01; cp<0,001). Beide Therapien (mit Kreatin oder Glibenklamid) erhöhen die Insulin- und C-Peptid-Konzentrationen nach 120 und 240 Minuten. Zusammenfassend konnte gezeigt werden, dass kurzfristige Therapien mit Kreatin oder Glibenklamid auf die Blutglukosekonzentration ähnliche Wirkungen entfalten.SummaryThis study compares the effects of glibenclamide and creatine in type II diabetics. In a 14-day symmetrically randomized crossover trial recently detected type II diabetics received either creatine (3 g) or glibenclamide (3.5 mg) for five successive days, followed by two days of washout, and crossover to the opposite treatment. Glucose, insulin, c-peptide, and creatine were measured. Creatine and glibenclamide decreased glucose concentrations vs. basal glucose [-15, 60, 90, 120, 180, and 240 min; mol/l]: 12.6±0.83 vs. 12.2±0.56 vs. 12.1±0.57, 15.3±0.63 vs. 13.5± 0.70a vs. 13.0±0.57a, 14.8±0.57 vs. 13.8±0.59avs. 13.4±0.46a, 14.6±0.61 vs. 12.3±0.49bvs. 12.4±0.61a, 12.8±0.76 vs. 10.0± 0.40cvs. 10.3±0.41c, and 11.4±0.67 vs. 8.3±0.40cvs. 8.5±0.36c; (ap<0.05; bp<0.01; cp<0.001 vs. basal glucose). Treatment with both creatine and glibenclamide increased insulin and c-peptide concentrations after 120 and 240 min (p<0.05 and p<0.01). At the doses applied short-term treatment with creatine and glibenclamide elicits similar glucose lowering effects.

Excitation-scanning hyperspectral imaging as a means to discriminate various tissues types

Little is currently known about the fluorescence excitation spectra of disparate tissues and how these spectra change with pathological state. Current imaging diagnostic techniques have limited capacity to investigate fluorescence excitation spectral characteristics. This study utilized excitation-scanning hyperspectral imaging to perform a comprehensive assessment of fluorescence spectral signatures of various tissues. Immediately following tissue harvest, a custom inverted microscope (TE-2000, Nikon Instruments) with Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) were used to acquire hyperspectral image data from each sample. Scans utilized excitation wavelengths from 340 nm to 550 nm in 5 nm increments. Hyperspectral images were analyzed with custom Matlab scripts including linear spectral unmixing (LSU), principal component analysis (PCA), and Gaussian mixture modeling (GMM). Spectra were examined for potential characteristic features such as consistent intensity peaks at specific wavelengths or intensity ratios among significant wavelengths. The resultant spectral features were conserved among tissues of similar molecular composition. Additionally, excitation spectra appear to be a mixture of pure endmembers with commonalities across tissues of varied molecular composition, potentially identifiable through GMM. These results suggest the presence of common autofluorescent molecules in most tissues and that excitationscanning hyperspectral imaging may serve as an approach for characterizing tissue composition as well as pathologic state. Future work will test the feasibility of excitation-scanning hyperspectral imaging as a contrast mode for discriminating normal and pathological tissues.

Feasibility for detection of autofluorescent signatures in rat organs using a novel excitation-scanning hyperspectral imaging system

The natural fluorescence (autofluorescence) of tissues has been noted as a biomarker for cancer for several decades. Autofluorescence contrast between tumors and healthy tissues has been of significant interest in endoscopy, leading to development of autofluorescence endoscopes capable of visualizing 2-3 fluorescence emission wavelengths to achieve maximal contrast. However, tumor detection with autofluorescence endoscopes is hindered by low fluorescence signal and limited quantitative information, resulting in prolonged endoscopic procedures, prohibitive acquisition times, and reduced specificity of detection. Our lab has designed a novel excitation-scanning hyperspectral imaging system with high fluorescence signal detection, low acquisition time, and enhanced spectral discrimination. In this study, we surveyed a comprehensive set of excised tissues to assess the feasibility of detecting tissue-specific pathologies using excitation-scanning. Fresh, untreated tissue specimens were imaged from 360 to 550 nm on an inverted fluorescence microscope equipped with a set of thin-film tunable filters (Semrock, A Unit of IDEX). Images were subdivided into training and test sets. Automated endmember extraction (ENVI 5.1, Exelis) with PCA identified endmembers within training images of autofluorescence. A spectral library was created from 9 endmembers. The library was used for identification of endmembers in test images. Our results suggest (1) spectral differentiation of multiple tissue types is possible using excitation scanning; (2) shared spectra between tissue types; and (3) the ability to identify unique morphological features in disparate tissues from shared autofluorescent components. Future work will focus on isolating specific molecular signatures present in tissue spectra, and elucidating the contribution of these signatures in pathologies.