Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions,but Not Blood Pressure,of Ace-/- Mice

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.     

The germinal isozyme of angiotensin-converting enzyme can substitute for the somatic isozyme in maintaining normal renal structure and functions.

The angiotensin-converting enzyme (ACE) gene encodes two structurally related isozymes, somatic ACE and germinal ACE, that are uniquely expressed in discrete locations in the body. The importance of ACE in these cell types was revealed by generating Ace -/- mice, which exhibit multiple abnormalities including renal structural defects and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by isozyme-specific and tissue-specific expression patterns, we have used a transgenic approach to develop mouse strains that express just one ACE isoform in the target tissue of Ace -/- mice. The mice described in this report produce germinal ACE in sperm and serum. These mice were as healthy as wild type mice, and the males were fertile. Interestingly, they had normal kidney structure, fluid homeostasis, and partially restored urine concentration despite having low blood pressure. This result demonstrated that circulating germinal ACE is sufficient for maintaining normal kidney structure and fluid homeostasis but insufficient for restoring blood pressure to normal levels.     

Angiotensin I converting enzyme in human intestine and kidney. Ultrastructural immunohistochemical localization

The localization of immunoreactive angiotensin I-converting enzyme (ACE) has been investigated at the optical and ultrastructural level with anti-human ACE antibodies in the human kidney and small intestine. In both tissues ACE was found in blood vessels and in extravascular situation in the absorptive epithelial cells of intestinal mucosa and renal proximal tubules. Ultrastructural immunohistochemistry showed that in intestinal and renal proximal tubular cells ACE was prominent in microvilli and brush borders. In the kidney ACE was also present on the basolateral part of the plasmalemmal membrane, where it may contribute to the regulation of angiotensin II-dependent absorption processes. Intracellular positivities were also observed inside the renal vascular endothelial and proximal tubular cell in endoplasmic reticulum and nuclear envelope reflecting the synthesis and the cellular processing of ACE. The intestinal microvascular endothelium was strongly labeled suggesting that the mesenteric circulation is an important site for the production of angiotensin II. Vascular endothelial ACE was also detected in the peritubular but not glomerular capillaries of the kidney.     

Angiotensin I converting enzyme in human intestine and kidney

Summary The localization of immunoreactive angiotensin I-converting enzyme (ACE) has been investigated at the optical and ultrastructural level with anti-human ACE antibodies in the human kidney and small intestine. In both tissues ACE was found in blood vessels and in extravascular situation in the absorptive epithelial cells of intestinal mucosa and renal proximal tubules. Ultrastructural immunohistochemistry showed that in intestinal and renal proximal tubular cells ACE was prominent in microvilli and brush borders. In the kidney ACE was also present on the basolateral part of the plasmalemmal membrane, where it may contribute to the regulation of angiotensin II-dependant absorption processes. Intracellular positivities were also observed inside the renal vascular endothelial and proximal tubular cell in endoplasmic reticulum and nuclear envelope reflecting the synthesis and the cellular processing of ACE. The intestinal microvascular endothelium was strongly labeled suggesting that the mesenteric circulation is an important site for the production of angiotensin II. Vascular endothelial ACE was also detected in the peritubular but not glomerular capillaries of the kidney.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

Summary The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Evidence for a functional intracellular angiotensin system in the proximal tubule of the kidney

ANG II is the most potent and important member of the classical renin-angiotensin system (RAS). ANG II, once considered to be an endocrine hormone, is now increasingly recognized to also play novel and important paracrine (cell-to-cell) and intracrine (intracellular) roles in cardiovascular and renal physiology and blood pressure regulation. Although an intracrine role of ANG II remains an issue of continuous debates and requires further confirmation, a great deal of research has recently been devoted to uncover the novel actions and elucidate underlying signaling mechanisms of the so-called intracellular ANG II in cardiovascular, neural, and renal systems. The purpose of this article is to provide a comprehensive review of the intracellular actions of ANG II, either administered directly into the cells or expressed as an intracellularly functional fusion protein, and its effects throughout a variety of target tissues susceptible to the impacts of an overactive ANG II, with a particular focus on the proximal tubules of the kidney. While continuously reaffirming the roles of extracellular or circulating ANG II in the proximal tubules, our review will focus on recent evidence obtained for the novel biological roles of intracellular ANG II in cultured proximal tubule cells in vitro and the potential physiological roles of intracellular ANG II in the regulation of proximal tubular reabsorption and blood pressure in rats and mice. It is our hope that the new knowledge on the roles of intracellular ANG II in proximal tubules will serve as a catalyst to stimulate further studies and debates in the field and to help us better understand how extracellular and intracellular ANG II acts independently or interacts with each other, to regulate proximal tubular transport and blood pressure in both physiological and diseased states.     

Transgenic mice over-expressing ET-1 in the endothelial cells develop systemic hypertension with altered vascular reactivity

Endothelin-1 (ET-1) is a potent vasoconstrictor involved in the regulation of vascular tone and implicated in hypertension. However, the role of small blood vessels endothelial ET-1 in hypertension remains unclear. The present study investigated the effect of chronic over-expression of endothelial ET-1 on arterial blood pressure and vascular reactivity using transgenic mice approach. Transgenic mice (TET-1) with endothelial ET-1 over-expression showed increased in ET-1 level in the endothelial cells of small pulmonary blood vessels. Although TET-1 mice appeared normal, they developed mild hypertension which was normalized by the ET(A) receptor (BQ123) but not by ET(B) receptor (BQ788) antagonist. Tail-cuff measurements showed a significant elevation of systolic and mean blood pressure in conscious TET-1 mice. The mice also exhibited left ventricular hypertrophy and left axis deviation in electrocardiogram, suggesting an increased peripheral resistance. The ionic concentrations in the urine and serum were normal in 8-week old TET-1 mice, indicating that the systemic hypertension was independent of renal function, although, higher serum urea levels suggested the occurrence of kidney dysfunction. The vascular reactivity of the aorta and the mesenteric artery was altered in the TET-1 mice indicating that chronic endothelial ET-1 up-regulation leads to vascular tone imbalance in both conduit and resistance arteries. These findings provide evidence for the role of spatial expression of ET-1 in the endothelium contributing to mild hypertension was mediated by ET(A) receptors. The results also suggest that chronic endothelial ET-1 over-expression affects both cardiac and vascular functions, which, at least in part, causes blood pressure elevation.     

Progressive Kidney Degeneration in Mice Lacking Tensin

Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, −/− mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell– matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell–matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.     

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

Localization of NBC-1 variants in human kidney and renal cell carcinoma

The Na(+)-HCO(3)(-) cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells.     

Expression of CD44 in kidney after acute ischemic injury in rats

De novo CD44 and ligand expression at wound margins accompanies cellular proliferation and migration that effect repair of injured mucosal and vascular endothelial tissues. To determine whether CD44 could play a role in recovery from acute ischemic renal injury, we characterized its renal expression and those of two of its ligands, hyaluronic acid and osteopontin. Although no expression is detectable in nonischemic kidneys, several mRNAs for CD44 are present within 1 day after injury. CD44 mRNA is expressed in proximal tubules undergoing repair. CD44 peptide is present in basal and lateral cell membranes. Hyaluronic acid is normally expressed in the interstitium of the renal papilla only. By 1 day postischemia, hyaluronic acid can be detected, in addition, in the interstitium surrounding regenerating tubules. Osteopontin, not normally expressed in the renal proximal tubule, is expressed in regenerating tubules by 3 days after induction of acute ischemic injury. Immunoreactive osteopontin peptide continues to be localized in those tubules still undergoing repair for as long as 7 days after the injury. Our data are consistent with a role for CD44-ligand interactions in the regenerating proximal tubule participating in the process of recovery after ischemic injury.     

Midkine is involved in neutrophil infiltration into the tubulointerstitium in ischemic renal injury.

Midkine (MK) is a multifunctional heparin-binding protein and promotes migration of neutrophils, macrophages, and neurons. In the normal mouse kidney, MK is expressed in the proximal tubules. After renal ischemic reperfusion injury, its expression in proximal tubules was increased. Immediate increase of MK expression was found when renal proximal tubular epithelial cells in culture were exposed to 5 mM H(2)O(2). Histologically defined tubulointerstitial damage was less severe in MK-deficient (Mdk(-/-)) than in wild-type (Mdk(+/+)) mice at 2 and 7 days after ischemic reperfusion injury. Within 2 days after ischemic injury, inflammatory leukocytes, of which neutrophils were the major population, were recruited to the tubulointerstitium. The numbers of infiltrating neutrophils and also macrophages were lower in Mdk(-/-) than in Mdk(+/+) mice. Induction of macrophage inflammatory protein-2 and macrophage chemotactic protein-1, chemokines for neutrophils and macrophages, respectively, were also suppressed in Mdk(-/-) mice. Furthermore, renal tubular epithelial cells in culture expressed macrophage inflammatory protein-2 in response to exogenous MK administration. These results suggested that MK enhances migration of inflammatory cells upon ischemic injury of the kidney directly and also through induction of chemokines, and contributes to the augmentation of ischemic tissue damage.     

Dysfunction of kidney endothelium after ischemia/reperfusion and its prevention by mitochondria-targeted antioxidant

One of the most important pathological consequences of renal ischemia/reperfusion (I/R) is kidney malfunctioning. I/R leads to oxidative stress, which affects not only nephron cells but also cells of the vascular wall, especially endothelium, resulting in its damage. Assessment of endothelial damage, its role in pathological changes in organ functioning, and approaches to normalization of endothelial and renal functions are vital problems that need to be resolved. The goal of this study was to examine functional and morphological impairments occurring in the endothelium of renal vessels after I/R and to explore the possibility of alleviation of the severity of these changes using mitochondria-targeted antioxidant 10-(6′-plastoquinonyl)decylrhodamine 19 (SkQR1). Here we demonstrate that 40-min ischemia with 10-min reperfusion results in a profound change in the structure of endothelial cells mitochondria, accompanied by vasoconstriction of renal blood vessels, reduced renal blood flow, and increased number of endothelial cells circulating in the blood. Permeability of the kidney vascular wall increased 48 h after I/R. Injection of SkQR1 improves recovery of renal blood flow and reduces vascular resistance of the kidney in the first minutes of reperfusion; it also reduces the severity of renal insufficiency and normalizes permeability of renal endothelium 48 h after I/R. In in vitro experiments, SkQR1 provided protection of endothelial cells from death provoked by oxygen–glucose deprivation. On the other hand, an inhibitor of NO-synthases, L-nitroarginine, abolished the positive effects of SkQR1 on hemodynamics and protection from renal failure. Thus, dysfunction and death of endothelial cells play an important role in the development of reperfusion injury of renal tissues. Our results indicate that the major pathogenic factors in the endothelial damage are oxidative stress and mitochondrial damage within endothelial cells, while mitochondria-targeted antioxidants could be an effective tool for the protection of tissue from negative effects of ischemia.     

Dimethylarginine dimethylaminohydrolase (DDAH): expression, regulation, and function in the cardiovascular and renal systems

Asymmetric (N(G),N(G))-dimethylarginine (ADMA) inhibits nitric oxide (NO) synthases (NOS). ADMA is a risk factor for endothelial dysfunction, cardiovascular mortality, and progression of chronic kidney disease. Two isoforms of dimethylarginine dimethylaminohydrolase (DDAH) metabolize ADMA. DDAH-1 is the predominant isoform in the proximal tubules of the kidney and in the liver. These organs extract ADMA from the circulation. DDAH-2 is the predominant isoform in the vasculature, where it is found in endothelial cells adjacent to the cell membrane and in intracellular vesicles and in vascular smooth muscle cells among the myofibrils and the nuclear envelope. In vivo gene silencing of DDAH-1 in the rat and DDAH +/- mice both have increased circulating ADMA, whereas gene silencing of DDAH-2 reduces vascular NO generation and endothelium-derived relaxation factor responses. DDAH-2 also is expressed in the kidney in the macula densa and distal nephron. Angiotensin type 1 receptor activation in kidneys reduces the expression of DDAH-1 but increases the expression of DDAH-2. This rapidly evolving evidence of isoform-specific distribution and regulation of DDAH expression in the kidney and blood vessels provides potential mechanisms for nephron site-specific regulation of NO production. In this review, the recent advances in the regulation and function of DDAH enzymes, their roles in the regulation of NO generation, and their possible contribution to endothelial dysfunction in patients with cardiovascular and kidney diseases are discussed.     

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys

Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohisto-chemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.     

Genetic evidence that lethality in angiotensinogen-deficient mice is due to loss of systemic but not renal angiotensinogen

Angiotensinogen (AGT)-deficient mice die shortly after birth presumably due to renal dysfunction caused by the presence of severe vascular and tubular lesions in the kidney. Because AGT is expressed in renal proximal tubule cells, we hypothesized that its loss may be the primary mediator of the lethal phenotype. We generated two models to test this hypothesis by breeding transgenic mice expressing human renin with mice expressing human AGT (hAGT) either systemically or kidney-specifically. We then bred double transgenic mice with AGT+/- mice, intercrossed the compound heterozygotes, and examined the offspring. We previously reported that the presence of the human renin and systemically expressed hAGT transgene complemented the lethality observed in AGT-/- mice. On the contrary, we show herein that the presence of the human renin and kidney-specific hAGT transgene cannot rescue lethality in AGT-/- mice. An analysis of newborns indicated that AGT-/- mice were born in normal numbers, and collection of dead 10-day old pups revealed an enrichment in AGT-/-. Importantly, we demonstrated that angiotensinogen protein and functional angiotensin II was generated in the kidney, and the kidney-specific transgene was temporally expressed during renal development similar to the endogenous AGT gene. These data strongly support the notion that the loss of systemic AGT, but not intrarenal AGT, is responsible for death in the AGT-/- mouse model. Taken together with our previous studies, we conclude that the intrarenal renin-angiotensin system located in the proximal tubule plays an important role in blood pressure regulation and may cause hypertension if overexpressed, but may not be required for continued development of the kidney after birth.     

Renal proximal tubule angiotensin AT1A receptors regulate blood pressure

All components of the renin angiotensin system necessary for ANG II generation and action have been reported to be present in renal proximal convoluted tubules. Given the close relationship between renal sodium handling and blood pressure regulation, we hypothesized that modulating the action of ANG II specifically in the renal proximal tubules would alter the chronic level of blood pressure. To test this, we used a proximal tubule-specific, androgen-dependent, promoter construct (KAP2) to generate mice with either overexpression of a constitutively active angiotensin type 1A receptor transgene or depletion of endogenous angiotensin type 1A receptors. Androgen administration to female transgenic mice caused a robust induction of the transgene in the kidney and increased baseline blood pressure. In the receptor-depleted mice, androgen administration to females resulted in a Cre recombinase-mediated deletion of angiotensin type 1A receptors in the proximal tubule and reduced blood pressure. In contrast to the changes observed at baseline, there was no difference in the blood pressure response to a pressor dose of ANG II in either experimental model. These data, from two separate mouse models, provide evidence that ANG II signaling via the type 1A receptor in the renal proximal tubule is a regulator of systemic blood pressure under baseline conditions.     

A novel podoplanin‐GFPCre mouse strain for gene deletion in lymphatic endothelial cells

The lymphatic vascular system is a one‐direction network of thin‐walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5’ regulatory region. Pdpn encodes a transmembrane mucin‐type O‐glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs.