Background: The mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.

We have developed a transgenic animal model to investigate the effects of overexpression of rat prorenin on the cardiovascular system. Two transgenic rat lines were generated in which rat prorenin expression was directed to the liver by a human ␣ 1-antitrypsin promoter. Liver-specific expression was confirmed by RNase protection assay. Plasma prorenin concentrations in transgenic rats were increased 400-fold in the males of both lines but were increased only two-to threefold in the females. Thus, transgene expression exhibited sexual dimorphism. Blood pressures were not significantly higher in transgenic rats than in nontransgenic controls. The ratio of heart weight to body weight was greater in male transgenic rats than in the nontransgenic controls.

The secretion of renin from granules stored in renal juxtaglomerular cells plays a key role in blood pressure homeostasis. The synthesis and release of renin and the extent of granulation is regulated by several mechanisms including signaling from the macula densa, neuronal input, and blood pressure. Through the use of a gene-targeting vector containing homology arms generated using the polymerase chain reaction, we have inactivated the Ren-1 d gene, one of two mouse genes encoding renin, and report that lack of renin-1 d results in altered morphology of the macula densa of the kidney distal tubule and complete absence of juxtaglomerular cell granulation. Furthermore, Ren-1 d؊/؊ mice exhibit sexually dimorphic hypotension. The altered growth morphology of the macula densa in Ren-1 d -null mice should provide a tool for the investigation of the JG cell-macula densa signaling. Furthermore, the current data indicate that expression of the Ren-1 d gene is a prerequisite for the formation of storage granules, even though the related protein renin-2 is present in these mice, suggesting that renin-1 d and renin-2 are secreted by distinct pathways in vivo.Renin (EC 3.4.23.15) is an aspartyl protease, which catalyzes the first step in the renin angiotensin system, the end product of which is the potent vasopressor peptide hormone, angiotensin II (AngII).1 This octapeptide acts to increase peripheral vascular resistance and promote salt and fluid retention in concert with the hormone aldosterone. Renin is synthesized principally in the kidney juxtaglomerular (JG) cells, a group of modified smooth muscle cells located at the distal end of the renal afferent arteriole of the glomerulus (1). JG cells are in close contact with the macula densa, a specialized plaque of epithelial cells of the kidney distal tubule, which signal to the renal arterioles to regulate glomerular filtration rate and the secretion of renin, in response to ionic concentration and flow rate in the distal tubule (2, 3), the so-called tubuloglomerular feedback loop. Except for the submandibular gland (SMG) of the mouse, the JG cells are the only site where prorenin, the inactive zymogen, is known to be converted to the active form of renin. SMG renin does not, however, make its way into the plasma in large quantities and is thought not to play a significant role in blood pressure regulation under normal circumstances (reviewed in Bing et al. (4)). The release of renin from JG cells is mediated by two pathways: regulated release of the mature, active renin from modified lysosomal storage granules and constitutive release of the inactive zymogen. While the regulated pathway of renin secretion is responsive to baroreceptor, neurogenic, and macula densa signals (5), the physiological significance of the constitutive secretion of prorenin is not understood, nor are the molecular pathways that link secretory signals to renin maturation and release. Clarification of the mechanisms underlying these processes will be crucial to our understanding of the control of ...

Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is coordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.

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The serine proteinase inhibitors (serpins) are a superfamily of proteins with a diverse set of functions, including the control of blood coagulation, complement activation, programmed cell death and development. The most abundant serpins in human plasma are alpha(1)-antitrypsin (AAT) and alpha(1)-antichymotrypsin (ACT). During inflammation, circulating levels can increase by up to 3-fold for the former and by 4-5-fold for the latter. The major site for increased synthesis is the liver. Other tissues, such as the lung, are also capable of synthesizing AAT and ACT, and expression can be increased by up to 100-fold by cytokines. There is a tissue-specific promoter for the liver, and alternative promoters for other tissues that express AAT. Basal AAT expression is regulated by the synergistic action of the tissue-specific transcription factors hepatocyte nuclear factors 1alpha and 4. An enhancer positioned approx. 1.2 kb from the end of the last exon in the 3' flanking sequence modulates cytokine-induced expression by interleukin-6 and oncostatin M. Microcell hybrid transfection studies have shown that a sequence containing 15 kb of 5' flanking sequence is sufficient to allow stable expression of AAT in a position-independent manner. There is probably a single promoter for ACT. Oncostatin M-inducible elements have been identified in the 5' flanking sequence approx. 100 bp upstream from the transcription initiation site, and a further interleukin-1-responsive enhancer has been identified approx. 13 kb upstream. The pathways for a humoral response are being mapped at high resolution.

Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2'-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6+/− heterozygotes but BrdU-LRCs were also present in Pax6+/− corneas. It seems likely that Pax6+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6+/− than WT mice. This implies that epithelial cell loss is higher in Pax6+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.