KCa3.1 Transgene Induction in Murine Intestinal Epithelium Causes Duodenal Chyme Accumulation and Impairs Duodenal Contractility.

Abstract: The epithelial intermediate-conductance calcium/calmodulin-regulated KCa3.1 channel is considered to be a regulator of intestine function by controlling chloride secretion and water/salt balance. Yet, little is known about the functional importance of KCa3.1 in the intestinal epithelium in vivo. Our objective was to determine the impact of epithelial-specific inducible overexpression of a KCa3.1 transgene (KCa3.1+) and of inducible suppression (KCa3.1-) on intestinal homeostasis and function in mice. KCa3.1 overexpression in the duodenal epithelium of doxycycline (DOX)-treated KCa3.1+ mice was 40-fold above the control levels. Overexpression caused an inflated duodenum and doubling of the chyme content. Histology showed conserved architecture of crypts, villi, and smooth muscle. Unaltered proliferating cell nuclear antigen (PCNA) immune reactivity and reduced amounts of terminal deoxynucleotide transferase mediated X-dUTP nick end labeling (TUNEL)-positive apoptotic cells in villi indicated lower epithelial turnover. Myography showed a reduction in the frequency of spontaneous propulsive muscle contractions with no change in amplitude. The amount of stool in the colon was increased and the frequency of colonic contractions was reduced in KCa3.1+ animals. Senicapoc treatment prevented the phenotype. Suppression of KCa3.1 in DOX-treated KCa3.1- mice caused no overt intestinal phenotype. In conclusion, inducible KCa3.1 overexpression alters intestinal functions by increasing the chyme content and reducing spontaneous contractions and epithelial apoptosis. Induction of epithelial KCa3.1 can play a mechanistic role in the process of adaptation of the intestine.

ImageJ-based semiautomatic method to analyze senescence in cell culture.

ß-Galactosidase accumulates in the lysosomes of senescent cells of certain tissues. Cell staining with X-gal is a common procedure to detect senescent cells in culture. However, the organelle nature of the staining makes automatic count impossible, requiring time-consuming manual counting or expensive alternative techniques such as flow cytometry to effectively determine the amount of stained cells. Here we present an analysis strategy for images of X-gal stained cells which can be implemented into a macro for the ImageJ software overcoming some of the drawbacks of computational analysis of organelle staining.

Macrophages in cardiac remodelling after myocardial infarction.

Myocardial infarction (MI), as a result of thrombosis or vascular occlusion, is the most prevalent cause of morbidity and mortality among all cardiovascular diseases. The devastating consequences of MI are compounded by the complexities of cellular functions involved in the initiation and resolution of early-onset inflammation and the longer-term effects related to scar formation. The resultant tissue damage can occur as early as 1 h after MI and activates inflammatory signalling pathways to elicit an immune response. Macrophages are one of the most active cell types during all stages after MI, including the cardioprotective, inflammatory and tissue repair phases. In this Review, we describe the phenotypes of cardiac macrophage involved in MI and their cardioprotective functions. A specific subset of macrophages called resident cardiac macrophages (RCMs) are derived from yolk sac progenitor cells and are maintained as a self-renewing population, although their numbers decrease with age. We explore sophisticated sequencing techniques that demonstrate the cardioprotective properties of this cardiac macrophage phenotype. Furthermore, we discuss the interactions between cardiac macrophages and other important cell types involved in the pathology and resolution of inflammation after MI. We summarize new and promising therapeutic approaches that target macrophage-mediated inflammation and the cardioprotective properties of RCMs after MI. Finally, we discuss future directions for the study of RCMs in MI and cardiovascular health in general.

Increased transmigration of intermediate monocytes associated with atherosclerotic burden in people with HIV on antiretroviral therapy.

This study evaluated the association between the transmigration of monocyte subpopulations that contributes to atherosclerosis development, along with surrogate biomarkers of inflammation and atherosclerosis, through carotid intima-media thickness (cIMT) measurements of 72 people with HIV (PWH) on suppressive antiretroviral therapy (ART). We found that the transmigration of intermediate monocytes was positively correlated with D-dimer and cIMT, suggesting that intermediate monocytes may have a greater propensity to promote cardiovascular disease (CVD) in PWH on ART.

Conditional KCa3.1-transgene induction in murine skin produces pruritic eczematous dermatitis with severe epidermal hyperplasia and hyperkeratosis.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-ß1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-ß1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target.

Pharmacological activation of TRPV4 produces immediate cell damage and induction of apoptosis in human melanoma cells and HaCaT keratinocytes.

BACKGROUND: TRPV4 channels are calcium-permeable cation channels that are activated by several physicochemical stimuli. Accordingly, TRPV4 channels have been implicated in the regulation of osmosensing, mechanotransduction, thermosensation, and epithelial/endothelial barrier functions. Whether TRPV4 is also mechanistically implicated in melanoma cell proliferation is not clear. Here, we hypothesized that TRPV4 is expressed in human melanoma and that pharmacological activation interferes with cell proliferation. METHODOLOGY/PRINCIPAL FINDINGS: TRPV4 functions were studied in melanoma cell lines (A375, SK-MEL-28, MKTBR), immortalized non-cancer keratinocytes (HaCaT), and murine 3T3 fibroblasts by patch-clamp, qRT-PCR, intracellular calcium measurements, cell proliferation, and flow cytometric assays of apoptosis and cell cycle. The selective TRPV4-activator, GSK1016790A, elicited non-selective cation currents with TRPV4-typical current-voltage-relationship in all cell lines. GSK1016790A-induced currents were blocked by the TRPV4-blocker, HC067047. TRPV4 mRNA expression was demonstrated by qRT-PCR. In A375 cells, TRPV4 activation was frequently paralleled by co-activation of calcium/calmodulin-regulated KCa3.1 channels. Light microscopy showed that TRPV4-activation produced rapid cellular disarrangement, nuclear densification, and detachment of a large fraction of all melanoma cell lines and HaCaT cells. TRPV4-activation induced apoptosis and drastically inhibited A375 and HaCaT proliferation that could be partially prevented by HC067047. CONCLUSIONS/SIGNIFICANCE: Our study showed that TRPV4 channels were functionally expressed in human melanoma cell lines and in human keratinocytes. Pharmacological TRPV4 activation in human melanoma cells and keratinocytes caused severe cellular disarrangement, necrosis and apoptosis. Pharmacological targeting of TRPV4 could be an alternative or adjuvant therapeutic strategy to treat melanoma progression and other proliferative skin disorders.

Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C.

The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression.