The life cycle of a capillary: Mechanisms of angiogenesis and rarefaction in microvascular physiology and pathologies.

Capillaries are the smallest blood vessels (<10 µm in diameter) in the body and their walls are lined by endothelial cells. These microvessels play a crucial role in nutrient and gas exchange between blood and tissues. Capillary endothelial cells also produce vasoactive molecules and initiate the electrical signals that underlie functional hyperemia and neurovascular coupling. Accordingly, capillary function and density are critical for all cell types to match blood flow to cellular activity. This begins with the process of angiogenesis, when new capillary blood vessels emerge from pre-existing vessels, and ends with rarefaction, the loss of these microvascular structures. This review explores the mechanisms behind these processes, emphasizing their roles in various microvascular diseases and their impact on surrounding cells in health and disease. We discuss recent work on the mechanisms controlling endothelial cell proliferation, migration, and tube formation that underlie angiogenesis under physiological and pathological conditions. The mechanisms underlying functional and anatomical rarefaction and the role of pericytes in this process are also discussed. Based on this work, a model is proposed in which the balance of angiogenic and rarefaction signaling pathways in a particular tissue match microvascular density to the metabolic demands of the surrounding cells. This negative feedback loop becomes disrupted during microvascular rarefaction: angiogenic mechanisms are blunted, reactive oxygen species accumulate, capillary function declines and eventually, capillaries disappear. This, we propose, forms the foundation of the reciprocal relationship between vascular density, blood flow, and metabolic needs and functionality of nearby cells.

TRPC1 channel clustering during store-operated Ca2+ entry in keratinocytes.

Skin is the largest organ in the human body with ∼95% of its surface made up of keratinocytes. These cells maintain a healthy skin barrier through regulated differentiation driven by Ca2+-transcriptional coupling. Many important skin conditions arise from disruption of this process although not all stages are fully understood. We know that elevated extracellular Ca2+ at the skin surface is detected by keratinocyte Gαq-coupled receptors that signal to empty endoplasmic reticulum Ca2+ stores. Orai channel store-operated Ca2+ entry (SOCE) and Ca2+ influx via "canonical" transient receptor potential (TRPC)-composed channels then activates transcription factors that drive differentiation. While STIM-mediated activation of Orai channels following store depletion is well defined, how TRPC channels are activated is less clear. Multiple modes of TRPC channel activation have been proposed, including 1) independent TRPC activation by STIM, 2) formation of Orai-TRPC-STIM complexes, and 3) the insertion of constitutively-active TRPC channels into the membrane during SOCE. To help distinguish between these models, we used high-resolution microscopy of intact keratinocyte (HaCaT) cells and immunogold transmission electron microscopy (TEM) of HaCaT plasma membrane sheets. Our data shows no evidence of significant insertion of Orai1 or TRPC subunits into the membrane during SOCE. Analysis of transmission electron microscopy data shows that during store-depletion and SOCE, Orai1 and TRPC subunits form separate membrane-localized clusters that migrate towards each other. This clustering of TRPC channel subunits in keratinocytes may support the formation of TRPC-STIM interactions at ER-plasma membrane junctions that are distinct from Orai-STIM junctions.

The formation of K V 2.1 macro-clusters is required for sex-specific differences in L-type Ca V 1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated Ca V 1.2 and K V 2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, K V 2.1 also plays a sex-specific role by promoting the clustering and activity of Ca V 1.2 channels. However, the impact of K V 2.1 protein organization on Ca V 1.2 function remains poorly understood. We discovered that K V 2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of K V 2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the K V 2.1 clustering site (K V 2.1 S590A ) eliminated K V 2.1 macro-clustering and sex-specific differences in Ca V 1.2 cluster size and activity. We propose that the degree of K V 2.1 clustering tunes Ca V 1.2 channel function in a sex-specific manner in arterial myocytes.

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes.

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

Store-operated calcium channels in skin.

The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca2+ entry (SOCE), whereby depletion of intracellular Ca2+ stores triggers Ca2+ influx across the plasma membrane, is central to the normal physiology of these cells and thus skin function. Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca2+ depletion and Orai channels that mediate Ca2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca2+-cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion.

Soluble adenylyl cyclase links Ca2+ entry to Ca2+/cAMP-response element binding protein (CREB) activation in vascular smooth muscle.

Ca2+-transcription coupling controls gene expression patterns that define vascular smooth muscle cell (VSMC) phenotype. Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca2+/HCO3--sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3',5'-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca2+/cAMP response element binding protein (CREB). Store-operated Ca2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca2+]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca2+, pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca2+-transcription coupling.

IL-17 Production from T Helper 17, Mucosal-Associated Invariant T, and γδ Cells in Tuberculosis Infection and Disease.

IL-17-producing cells have been shown to be important in the early stages of Mycobacterium tuberculosis (Mtb) infection in animal models. However, there are very little data on the role of IL-17 in human studies of tuberculosis (TB). We recruited TB patients and their highly exposed contacts who were further categorized based on results from an IFN-γ-release assay (IGRA): (1) IGRA positive (IGRA+) at recruitment (latently TB infected), (2) IGRA negative (IGRA-) at recruitment and 6 months [non-converters (NC)], and (3) IGRA- at recruitment and IGRA+ at 6 months (converters). Whole blood was stimulated with mycobacterial antigens and analyzed using T helper (Th) 17 multiplex cytokine assays. Th17, Vγ9Vδ2+, and CD161++Vα7.2+ mucosal-associated invariant T (MAIT) cells were analyzed by flow cytometry. The majority of IL-17 was produced by CD26+CD4+ Th17 cells (median 71%) followed by γδ T cells (6.4%) and MAIT cells (5.8%). TB patients had a significantly lower proportion of Th17 cells and CD4+CD161+Vα7.2+ cells producing both IL-17 and IFN-γ compared to LTBI subjects. IGRA NC had significantly lower levels of CD26-CD4+ and CD8+ MAIT cells producing IL-17 compared to IGRA C but had significantly higher levels of IL-17A, IL-17F, IL-21, and IL-23 in ESAT-6/CFP-10-stimulated supernatants compared to IGRA C. These data provide new insights into the role of IL-17 and IL-17-producing cells at three key stages of the Mtb infection spectrum.