Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF

Most endocrine hormones are produced in tissues and organs with permeable microvessels that may provide an excess of hormones to be transported by the blood circulation to the distal target organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence of other angiogenic factors. We provide evidence that leptin-induced new blood vessels are fenestrated. Under physiological conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient ob/ob mice. Thus, leptin plays a critical role in the maintenance and regulation of vascular fenestrations in the adipose tissue. Leptin induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate that leptin has another new function—the increase of vascular permeability.

Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

Similarities and dissimilarities of phage genomes.

Genomic similarities and contrasts are investigated in a collection of 23 bacteriophages, including phages with temperate, lytic, and parasitic life histories, with varied sequence organizations and with different hosts and with different morphologies. Comparisons use relative abundances of di-, tri-, and tetranucleotides from entire genomes. We highlight several specific findings. (i) As previously shown for cellular genomes, each viral genome has a distinctive signature of short oligonucleotide abundances that pervade the entire genome and distinguish it from other genomes. (ii) The enteric temperate double-stranded (ds) phages, like enterobacteria, exhibit significantly high relative abundances of GpC = GC and significantly low values of TA, but no such extremes exist in ds lytic phages. (iii) The tetranucleotide CTAG is of statistically low relative abundance in most phages. (iv) The DAM methylase site GATC is of statistically low relative abundance in most phages, but not in P1. This difference may relate to controls on replication (e.g., actions of the host SeqA gene product) and to MutH cleavage potential of the Escherichia coli DAM mismatch repair system. (v) The enteric temperate dsDNA phages form a coherent group: they are relatively close to each other and to their bacteria] hosts in average differences of dinucleotide relative abundance values. By contrast, the lytic dsDNA phages do not form a coherent group. This difference may come about because the temperate phages acquire more sequence characteristics of the host because they use the host replication and repair machinery, whereas the analyzed lytic phages are replicated by their own machinery. (vi) The nonenteric temperate phages with mycoplasmal and mycobacterial hosts are relatively close to their respective hosts and relatively distant from any of the enteric hosts and from the other phages. (vii) The single-stranded RNA phages have dinucleotide relative abundance values closest to those for random sequences, presumably attributable to the mutation rates of RNA phages being much greater than those of DNA phages.

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea.

Here we show that the mature cochlear neurons are a rich source of acidic fibroblast growth factor (aFGF), which is expressed in the neuronal circuitry consisting of afferent and efferent innervation. The site of action of neuronal aFGF is likely to reside in the organ of Corti, where one of the four known FGF receptor (FGFR) tyrosine kinases--namely, FGFR-3 mRNA--is expressed. Following acoustic overstimulation, known to cause damage to the organ of Corti, a rapid up-regulation of FGFR-3 is evident in this sensory epithelium, at both mRNA and protein levels. The present results provide in vivo evidence for aFGF being a sensory neuron-derived, anterogradely transported factor that may exert trophic effects on a peripheral target tissue. In this sensory system, aFGF, rather than being a neurotrophic factor, seems to promote maintenance of the integrity of the organ of Corti. In addition, aFGF, released from the traumatized nerve endings, may be one of the first signals initiating protective recovery and repair processes following damaging auditory stimuli.

Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.

The goal of this study was to determine whether sphingosine and ceramide, second messengers derived from sphingolipid breakdown, alter kidney proximal tubular cell viability and their adaptive responses to further damage. Adult human kidney proximal tubular (HK-2) cells were cultured for 0-20 hr in the presence or absence of sphingosine, sphingosine metabolites (sphingosine 1-phosphate, dimethylsphingosine), or C2, C8, or C16 ceramide. Acute cell injury was assessed by vital dye exclusion and tetrazolium dye transport. Their subsequent impact on superimposed ATP depletion/Ca2+ ionophore-induced damage was also assessed. Sphingosine (> or = 10 microM), sphingosine 1-phosphate, dimethylsphingosine, and selected ceramides (C2 and C8, but not C16) each induced rapid, dose-dependent cytotoxicity. This occurred in the absence of DNA laddering or morphologic changes of apoptosis, suggesting a necrotic form of cell death. Prolonged exposure (20 hr) to subtoxic sphingosine doses (< or = 7.5 microM) induced substantial cytoresistance to superimposed ATP depletion/Ca2+ ionophore-mediated damage. Conversely, neither short-term sphingosine treatment (< or = 8.5 hr) nor 20-hr exposures to any of the above sphingosine/ceramide derivatives/metabolites or various free fatty acids reproduced this effect. Sphingosine-induced cytoresistance was dissociated from the extent of cytosolic Ca2+ loading (indo-1 fluorescence), indicating a direct increase in cell resistance to attack. We conclude that sphingosine can exert dual effects on proximal renal tubular viability: in high concentrations it induces cell necrosis, whereas in low doses it initiates a cytoresistant state. These results could be reproduced in human foreskin fibroblasts, suggesting broad-based relevance to the area of acute cell injury and repair.

The causes and prevention of cancer.

Epidemiological evidence indicates that avoidance of smoking, increased consumption of fruits and vegetables, and control of infections will have a major effect on reducing rates of cancer. Other factors include avoidance of intense sun exposure, increases in physical activity, and reduction of alcohol consumption and possibly red meat. A substantial reduction in breast cancer is likely to require modification of sex hormone levels, and development of practical methods for doing so is a high research priority. Resolution of the potential protective roles of specific antioxidants and other constituents of fruits and vegetables deserves major attention. Mechanistic studies of carcinogenesis indicate an important role of endogenous oxidative damage to DNA that is balanced by elaborate defense and repair processes. Also key is the rate of cell division, which is influenced by hormones, growth, cytotoxicity, and inflammation, as this determines the probability of converting DNA lesions to mutations. These mechanisms may underlie many epidemiologic observations.

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.