CaMK4 is a downstream effector of the α1G T-type calcium channel to determine the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α1G-subtype of voltage-gated T-type Ca2+ channel. We have previously revealed that the α1G channel functions as a background Ca2+ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α1G-mediated Ca2+ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α1G-mediated Ca2+ entry to Akt phosphorylation remain incompletely understood. In this study, we demonstrate that Ca2+/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α1G-mediated Ca2+ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α1G/Ca2+/CaMK4 is independent of the α1G/Ca2+/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α1G-CaMK4 signaling complex that regulates the Ca2+-dominated angiogenic potential in PMVECs.

α1G T-type calcium channel determines the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic "network formation" capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell's angiogenic potential via a PI3K-Akt signaling pathway.

A perpetual switching system in pulmonary capillaries.

Of the 300 billion capillaries in the human lung, a small fraction meet normal oxygen requirements at rest, with the remainder forming a large reserve. The maximum oxygen demands of the acute stress response require that the reserve capillaries are rapidly recruited. To remain primed for emergencies, the normal cardiac output must be parceled throughout the capillary bed to maintain low opening pressures. The flow-distributing system requires complex switching. Because the pulmonary microcirculation contains contractile machinery, one hypothesis posits an active switching system. The opposing hypothesis is based on passive switching that requires no regulation. Both hypotheses were tested ex vivo in canine lung lobes. The lobes were perfused first with autologous blood, and capillary switching patterns were recorded by videomicroscopy. Next, the vasculature of the lobes was saline flushed, fixed by glutaraldehyde perfusion, flushed again, and then reperfused with the original, unfixed blood. Flow patterns through the same capillaries were recorded again. The 16-min-long videos were divided into 4-s increments. Each capillary segment was recorded as being perfused if at least one red blood cell crossed the entire segment. Otherwise it was recorded as unperfused. These binary measurements were made manually for each segment during every 4 s throughout the 16-min recordings of the fresh and fixed capillaries (>60,000 measurements). Unexpectedly, the switching patterns did not change after fixation. We conclude that the pulmonary capillaries can remain primed for emergencies without requiring regulation: no detectors, no feedback loops, and no effectors-a rare system in biology. NEW & NOTEWORTHY The fluctuating flow patterns of red blood cells within the pulmonary capillary networks have been assumed to be actively controlled within the pulmonary microcirculation. Here we show that the capillary flow switching patterns in the same network are the same whether the lungs are fresh or fixed. This unexpected observation can be successfully explained by a new model of pulmonary capillary flow based on chaos theory and fractal mathematics.

Lectin-Based Characterization of Vascular Cell Microparticle Glycocalyx.

Microparticles (MPs) are released constitutively and from activated cells. MPs play significant roles in vascular homeostasis, injury, and as biomarkers. The unique glycocalyx on the membrane of cells has frequently been exploited to identify specific cell types, however the glycocalyx of the MPs has yet to be defined. Thus, we sought to determine whether MPs, released both constitutively and during injury, from vascular cells have a glycocalyx matching those of the parental cell type to provide information on MP origin. For these studies we used rat pulmonary microvascular and artery endothelium, pulmonary smooth muscle, and aortic endothelial cells. MPs were collected from healthy or cigarette smoke injured cells and analyzed with a panel of lectins for specific glycocalyx linkages. Intriguingly, we determined that the MPs released either constitutively or stimulated by CSE injury did not express the same glycocalyx of the parent cells. Further, the glycocalyx was not unique to any of the specific cell types studied. These data suggest that MPs from both normal and healthy vascular cells do not share the parental cell glycocalyx makeup.

A unique pulmonary microvascular endothelial cell niche revealed by Weibel-Palade bodies and Griffonia simplicifolia.

Pulmonary endothelium displays considerable heterogeneity along the vascular axis, from arteries to capillaries to veins. Griffonia simplicifolia is a lectin that recognizes pulmonary microvascular endothelium with preference over extra-alveolar endothelium in both arteries and veins, yet the precise vascular location where this phenotypic shift occurs is poorly resolved. We gelatin-filled the circulation and agarose-loaded the airways and then labeled the lung with Griffonia lectin to enable visualization of the endothelial transition zone. Endothelium in vessels with internal diameters less than 38 µm were uniformly Griffonia positive, whereas vessels with internal diameters greater than 60 µm were always Griffonia negative. Two populations of endothelium were identified in vessels ranging from 38 to 60 µm in diameter, including some that were positive and others that were negative for binding to G. simplicifolia. To better resolve this endothelial transition zone, we performed morphology studies to measure the distribution of Weibel-Palade bodies (WPbs), since WPbs are present in conduit vessel endothelium and absent in capillary endothelium. WPbs were found in endothelium with vascular dimensions as small as 18 µm in diameter but were not found in capillaries. Thus, we identify with precision that the endothelial phenotype transition from a cell that does not interact with Griffonia lectin to one that does occurs in blood vessels with internal diameters of approximately 38 µm, and we reveal an unappreciated vascular zone, between 18 and 38 µm in diameter, where endothelium both is Griffonia positive and possesses WPbs.

The cancer paradigm of severe pulmonary arterial hypertension.

The plexiform lesions of severe pulmonary arterial hypertension (PAH) are similar in histologic appearance, whether the disease is idiopathic or secondary. Both forms of the disease show actively proliferating endothelial cells without evidence of apoptosis. Here, we discuss the pathobiology of the atypical, angioproliferative endothelial cells in severe PAH. The concept of the endothelial cell as a "quasi-malignant" cell provides a new framework for antiproliferative, antiangiogenic therapy in severe PAH.

Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium.

Pulmonary microvascular endothelial cells (PMVECs) are enriched with progenitor cells that underlie their rapid proliferation and vasculogenic capacity. However, the molecular basis for such an enhanced growth potential is unknown. Nucleosome assembly protein-1 (NAP1), and its related family of proteins, have been incriminated in control of cell growth in a range of species. We therefore sought to determine whether NAP1 contributes to the rapid proliferation and vasculogenesis that is observed in PMVECs. NAP1 was expressed at a high level in two fast-growing cell types, including PMVECs and resident microvascular endothelial progenitor cells that were selected from PMVECs, whereas it was expressed at a low level in slow-growing pulmonary artery endothelial cells (PAECs). Heterologous NAP1 expression increased the growth potential of PAECs, whereas inhibiting NAP1 expression reduced the growth potential of PMVECs. Despite its impact on endothelial cell growth, NAP1 did not influence the expression of endothelial cell-selective markers (PECAM-1, VE-cadherin, von Willebrand factor), and it did not influence cell type-specific lectin binding criterion; PMVECs interact with Griffonia simplicifolia lectin, whereas PAECs interact with Helix pomatia lectin. PMVECs possess a higher basal transelectrical resistance than do PAECs, indicative of their more restrictive barrier property. Changing NAP1 expression did not normalize this basal barrier function between PMVECs and PAECs. To examine whether the growth-promoting actions of NAP1 influence blood vessel formation, endothelial cells were mixed into Matrigel and subcutaneously implanted. PMVECs generated eightfold more blood vessels than did PAECs over a 10-day time course. Heterologous NAP1 expression in PAECs increased the number of blood vessels formed by this cell type, where blood vessel growth approached that seen with PMVECs. Thus, our findings indicate that NAP1 functions as an important regulator of the proliferative and vasculogenic endothelial cell phenotype without globally impacting endothelial cell phenotype specification.

Microtubule motors regulate ISOC activation necessary to increase endothelial cell permeability.

Calcium store depletion activates multiple ion channels, including calcium-selective and nonselective channels. Endothelial cells express TRPC1 and TRPC4 proteins that contribute to a calcium-selective store-operated current, I(SOC). Whereas thapsigargin activates the I(SOC) in pulmonary artery endothelial cells (PAECs), it does not activate I(SOC) in pulmonary microvascular endothelial cells (PMVECs), despite inducing a significant rise in global cytosolic calcium. Endoplasmic reticulum exhibits retrograde distribution in PMVECs when compared with PAECs. We therefore sought to determine whether endoplasmic reticulum-to-plasma membrane coupling represents an important determinant of I(SOC) activation in PAECs and PMVECs. Endoplasmic reticulum organization is controlled by microtubules, because nocodozole induced microtubule disassembly and caused retrograde endoplasmic reticulum collapse in PMVECs. In PMVECs, rolipram treatment produced anterograde endoplasmic reticulum distribution and revealed a thapsigargin-activated I(SOC) that was abolished by nocodozole and taxol. Microtubule motors control organelle distribution along microtubule tracks, with the dynein motor causing retrograde movement and the kinesin motor causing anterograde movement. Dynamitin expression reduces dynein motor function inducing anterograde endoplasmic reticulum transport, which allows for direct activation of I(SOC) by thapsigargin in PMVECs. In contrast, expression of dominant negative kinesin light chain reduces kinesin motor function and induces retrograde endoplasmic reticulum transport; dominant negative kinesin light chain expression prevented the direct activation of I(SOC) by thapsigargin in PAECs. I(SOC) activation is an important step leading to disruption of cell-cell adhesion and increased macromolecular permeability. Thus, microtubule motor function plays an essential role in activating cytosolic calcium transitions through the membrane I(SOC) channel leading to endothelial barrier disruption.

An intriguing case: malignant mesothelioma presenting as inguinal lymph node metastases.

A 56-year-old woman presented with a right inguinal mass. Biopsy revealed multiple lymph nodes involved with papillary and gland-like structures extending into the surrounding fibroadipose tissue. There was no history of carcinoma or other malignancy. Ultrastructural findings included long microvilli, desmosomes, and tonofilament bundles, indicating metastatic malignant mesothelioma. Malignant mesothelioma only rarely presents as a lymph node metastasis, and electron microscopy is very useful in establishing this diagnosis.

Effect of cyclosporin pharmacokinetics on renal allograft outcome in African-Americans.

African-Americans (A-As) experience inferior outcome after transplantation compared with other ethnic groups. Bioavailability of cyclosporin (CsA) has been implicated as a possible contributing factor. This paper describes the outcome of 32 A-A recipients of de novo renal allograft who received CsA-based triple immunotherapy according to individual pharmacokinetic profiles. Patients received CsA-microemulsion q 12 h, dosed initially at 3.5 mg/kg (8 am) and 3.0 mg/kg (8 pm). The am and pm doses were independently adjusted to achieve a 12-h area under the concentration-time curve (AUC0-12) of 6600-7200 nghr/mL and morning trough level (C0) of 250-325 ng/mL, respectively. Mean age was 43 +/- 12 yr, 37% (12) female. Mean AUC0-12 in 1 wk, 1, 3, 6, and 12 months were 7810 +/- 1880 nghr/mL, 9057 +/- 2097 nghr/mL, 7674 +/- 1912 nghr/mL, 7132 +/- 2040 nghr/mL, and 6503 +/- 1410 ngl/h with corresponding C0 of 301 +/- 79 ng/mL, 316 +/- 66 ng/mL, 275 +/- 59 ng/mL, 273 +/- 66 ng/mL, and 224 +/- 49 ng/mL, respectively. Acute rejection occurred in two patients (6%) 1 yr after transplantation. Prospective use of CsA pharmocokinetic profiles improves renal allograft outcome in A-As.

Titanium particles identified by energy-dispersive X-ray microanalysis within the lungs of a painter at autopsy.

A 72-year-old male painter, who complained of his "lungs burning" for 2 weeks, died suddenly. Autopsy examination revealed severe coronary atherosclerosis with plaque rupture as the cause of death. Examination of the lungs revealed emphysema, interstitial fibrosis, and multinucleated giant cells with intra- and extracellular brown-black, crystalline, polarizable foreign material. Energy-dispersive X-ray microanalysis showed the material to contain titanium, aluminum, silicon, and iron. An increased incidence of respiratory disease has been reported in professional painters. Titanium is widely used as a pigment in the manufacturing of commercial paints. Cases of pneumoconiosis and alveolar proteinosis have been described in painters in which analysis of lung tissue revealed increased levels of titanium. This case is presented as an example of a rarely reported phenomenon, which may have clinical implications for evaluation and management of lung disease in painters.

The gill arch of the striped bass (Morone saxatilis). IV. Alterations in the ultrastructure of chloride cell apical crypts and chloride efflux following exposure to seawater.

Osmotically induced alterations in the ultrastructure of the apical crypts of chloride cells and changes in chloride efflux were studied in striped bass (Morone saxatilis). Striped bass were divided into three groups: fish adapted to freshwater, fish transferred directly from freshwater to 100% seawater (3% salt, w/v) for 24 hr or less, and fish adapted to 100% seawater for 7 days or more. Transmission electron microscopy studies revealed multicellular complexes of cells in both freshwater- and seawater-adapted fish. Cytoplasmic indigitations between cells in the complex were more numerous in seawateradapted bass. Scanning electron microscopy studies showed that the apical extensions in freshwater fish were uniform in size. Changes in ultrastructure and chloride efflux were observed within 3 hr after transfer to seawater. Initially the apical extensions of chloride cells become longer, more prominent, and branched. After 7 days in seawater some of the apical crypts develop into a deeper "pit" structure, while others remain like those of freshwater fish. An increase in the number of apical crypts is measured by 14 days after transfer. Chloride efflux increases to five times freshwater values after 24 hr and 17 times freshwater values after 7 days in seawater. Mitochondrial density is not significantly different between freshwater and seawater fish (7 or more days). The response of chloride cell apical crypts is not an all-or-none phenomenon as observed in other species. Striped bass are able to increase chloride efflux when osmotically stressed with little ultrastructural alteration.