Airway hillocks are injury-resistant reservoirs of unique plastic stem cells.

Airway hillocks are stratified epithelial structures of unknown function1. Hillocks persist for months and have a unique population of basal stem cells that express genes associated with barrier function and cell adhesion. Hillock basal stem cells continually replenish overlying squamous barrier cells. They exhibit dramatically higher turnover than the abundant, largely quiescent classic pseudostratified airway epithelium. Hillocks resist a remarkably broad spectrum of injuries, including toxins, infection, acid and physical injury because hillock squamous cells shield underlying hillock basal stem cells from injury. Hillock basal stem cells are capable of massive clonal expansion that is sufficient to resurface denuded airway, and eventually regenerate normal airway epithelium with each of its six component cell types. Hillock basal stem cells preferentially stratify and keratinize in the setting of retinoic acid signalling inhibition, a known cause of squamous metaplasia2,3. Here we show that mouse hillock expansion is the cause of vitamin A deficiency-induced squamous metaplasia. Finally, we identify human hillocks whose basal stem cells generate functional squamous barrier structures in culture. The existence of hillocks reframes our understanding of airway epithelial regeneration. Furthermore, we show that hillocks are one origin of 'squamous metaplasia', which is long thought to be a precursor of lung cancer.

A nanoparticle probe for the imaging of autophagic flux in live mice via magnetic resonance and near-infrared fluorescence.

Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Adhesion-GPCR Gpr116 (ADGRF5) expression inhibits renal acid secretion.

The diversity and near universal expression of G protein-coupled receptors (GPCR) reflects their involvement in most physiological processes. The GPCR superfamily is the largest in the human genome, and GPCRs are common pharmaceutical targets. Therefore, uncovering the function of understudied GPCRs provides a wealth of untapped therapeutic potential. We previously identified an adhesion-class GPCR, Gpr116, as one of the most abundant GPCRs in the kidney. Here, we show that Gpr116 is highly expressed in specialized acid-secreting A-intercalated cells (A-ICs) in the kidney using both imaging and functional studies, and we demonstrate in situ receptor activation using a synthetic agonist peptide unique to Gpr116. Kidney-specific knockout (KO) of Gpr116 caused a significant reduction in urine pH (i.e., acidification) accompanied by an increase in blood pH and a decrease in pCO2 compared to WT littermates. Additionally, immunogold electron microscopy shows a greater accumulation of V-ATPase proton pumps at the apical surface of A-ICs in KO mice compared to controls. Furthermore, pretreatment of split-open collecting ducts with the synthetic agonist peptide significantly inhibits proton flux in ICs. These data suggest a tonic inhibitory role for Gpr116 in the regulation of V-ATPase trafficking and urinary acidification. Thus, the absence of Gpr116 results in a primary excretion of acid in KO mouse urine, leading to mild metabolic alkalosis ("renal tubular alkalosis"). In conclusion, we have uncovered a significant role for Gpr116 in kidney physiology, which may further inform studies in other organ systems that express this GPCR, such as the lung, testes, and small intestine.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

BACKGROUND: Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. METHODS: Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. RESULTS: Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. CONCLUSIONS: A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Amyloid-ß42/40 ratio drives tau pathology in 3D human neural cell culture models of Alzheimer's disease.

The relationship between amyloid-ß (Aß) species and tau pathology in Alzheimer's disease (AD) is not fully understood. Here, we provide direct evidence that Aß42/40 ratio, not total Aß level, plays a critical role in inducing neurofibrillary tangles (NTFs) in human neurons. Using 3D-differentiated clonal human neural progenitor cells (hNPCs) expressing varying levels of amyloid ß precursor protein (APP) and presenilin 1 (PS1) with AD mutations, we show that pathogenic tau accumulation and aggregation are tightly correlated with Aß42/40 ratio. Roles of Aß42/40 ratio on tau pathology are also confirmed with APP transmembrane domain (TMD) mutant hNPCs, which display differential Aß42/40 ratios without mutant PS1. Moreover, naïve hNPCs co-cultured with APP TMD I45F (high Aß42/40) cells, not with I47F cells (low Aß42/40), develop robust tau pathology in a 3D non-cell autonomous cell culture system. These results emphasize the importance of reducing the Aß42/40 ratio in AD therapy.

Cosmc-dependent mucin-type O-linked glycosylation is essential for podocyte function.

Mucin-type O-linked glycosylation, a posttranslational modification affecting the stability and biophysical characteristics of proteins, requires C1GalT1 (T synthase) and its obligate, X-linked chaperone Cosmc. Hypomorphic C1GalT1 mutations cause renal failure via not yet established mechanisms. We hypothesize that impaired Cosmc-dependent O-glycosylation in podocytes is sufficient to cause disease. Podocyte-specific Cosmc knockout mice were generated and phenotyped to test this hypothesis. Female heterozygous mice displaying mosaic inactivation of Cosmc in podocytes due to random X-linked inactivation were also examined. Mice with podocyte-specific Cosmc deletion develop profound albuminuria, foot process effacement, glomerular sclerosis, progressive renal failure, and impaired survival. Glomerular transcriptome analysis reveals early changes in cell adhesion, extracellular matrix organization, and chemokine-mediated signaling pathways, coupled with podocyte loss. Expression of the O-glycoprotein podoplanin was lost, while Tn antigen, representing immature O-glycans, was most abundantly found on podocalyxin. In contrast to hemizygous male and homozygous female animals, heterozygous female mosaic animals developed only mild albuminuria, focal foot process effacement, and nonprogressive kidney disease. Ultrastructurally, Cosmc-deficient podocytes formed Tn antigen-positive foot processes interdigitating with those of normal podocytes but not with other Cosmc-deficient cells. This suggests a cell nonautonomous mechanism for mucin-type O-glycoproteins in maintaining podocyte function. In summary, our findings demonstrated an essential and likely cell nonautonomous role for mucin-type O-glycosylation for podocyte function.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Targeted deletion of the Ncoa7 gene results in incomplete distal renal tubular acidosis in mice.

We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.

Extracellular Adenosine Stimulates Vacuolar ATPase-Dependent Proton Secretion in Medullary Intercalated Cells.

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H+-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H+ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration-approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)-dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway-dependent mechanism to induce V-ATPase-dependent H+ secretion.

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Deletion of ß1-integrin in collecting duct principal cells leads to tubular injury and renal medullary fibrosis.

The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of ß1-integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of ß1-integrin specifically in ICs and PCs, respectively, did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of ß1-integrin in CD cells, specifically in the PCs. We conditionally deleted ß1-integrin in mouse CD PCs using a specific aquaporin-2 (AQP2) promoter Cre-LoxP system. The resulting mutant mice, ß-1f/fAQP2-Cre+, had lower body weight, failed to thrive, and died around 8-12 wk. Their CD tubules were dilated, and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of transforming growth factor-ß (TGF-ß)-induced protein, fibronectin, and TGF-ß receptor-1 mRNAs and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-ß signaling pathway. Therefore, our data reveal that normal expression of ß1-integrin in PCs is a critical determinant of CD structural and functional integrity and further support the previously reported critical role of ß1-integrin in the development and/or maintenance of the CD structure and function.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System.

ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

Multiple wall in-folds sub-divide single segments during capillary regression in hyperoxic acute lung injury.

The present study provides further insight into the structural processes that remodel pulmonary capillaries in the injured adult lung. Early in hyperoxia acute lung injury (HALI), many sub-dividing segments are present throughout the capillary network before segment occlusion and loss predominate and capillary density decreases later in the period. A second segment sub-division triggered in regenerating capillaries after air breathing (post-HALI) demonstrates a similar mechanism of organization at a time of contrasting change in the capillary density. As we have previously reported, the process of segment sub-division includes in-folding of the endothelial-epithelial surface (alveolar-capillary membrane) to form inter-luminal structures (ILSs) and loops, with loop separation increasing segment number. Unexpectedly, the findings support remodeling of the capillary density by wall in-folding in acute lung injury, demonstrating a similar mechanism in capillary regression as well as in regeneration in the adult lung.

Imaging circulating monocytic cells fusing to endothelial cells in acutely injured and regenerating capillaries.

The present studies focus on monocytic circulating cells (CCs) interacting with the endothelial cells of pulmonary capillaries in acute lung injury. The CCs are further defined into sub-sets based on their structural profiles, i.e. CC(1-3). They are shown to move into close apposition to adjacent capillary endothelium and to fuse to endothelial plasmalemmal membranes. Similarly, CCs are seen to fuse to the endothelial cells of regenerating capillaries after injury. Immunogold labeling studies demonstrate that CCs express a mediator promoting endothelial cell migration, proliferation and stability, i.e. VEGF, further supporting the potential of a paracrine interaction between the fusing cells, while the expression of CXCR4 by CCs, and of SDF-1α by adjacent endothelial cells, demonstrates a mechanism for retention of these cells at the capillary surface. Myeloid VEGF-R2(+)CD11b(+) precursors and PDGF-Rß(+) expressing cells are identified within the CC population. The findings establish that, by fusing to endothelial cells, the monocytic CC population studied has the potential to promote capillary surface stability/integrity through a paracrine mechanism.

Mechanisms of growth of a pulmonary capillary network in adult lung.

The present study provides new insight into structural processes remodeling pulmonary capillaries in adult lung. The data highlight mechanisms underlying the expansion and increased density of capillary segments on return to air breathing (FiO2 0.21) after injury in high oxygen (FiO2 0.75). As segments expand and increase in number, endothelial cells extend their processes to bridge the lumen and support the walls of developing interluminal structures (ILSs); endothelial-epithelial surfaces infold as a single unit (sheet) into the lumen, increasing the length of each surface and subdividing segments by loop formation and by the formation of ILSs; segments further increase in number as lumen subdivision proceeds by intussusceptive microvascular growth (IMG).

Alveolar oxygen tension and angio-architecture of the distal adult lung.

The present study demonstrates the fine structure of pulmonary capillaries first injured and then undergoing growth in response to a change in the ambient alveolar oxygen tension. Breathing a high fraction of inspired oxygen (FiO2 0.75) triggers restriction by endothelial cell injury and effacement leading to segment narrowing and shortening and segment loss as demonstrated by a fall in density. Subsequently, breathing a relatively low fraction (FiO2 0.21) triggers capillary assembly (angiogenesis), which reverses the changes. The data underscore the structural reprogramming (reduction and restoration) of pulmonary capillaries in response to significant shifts in oxygen tension.