Overexpression of nerve growth factor in epidermis disrupts the distribution and properties of sympathetic innervation in footpads

dwarf4 (dwf4) mutants of Arabidopsis display a dwarfed phenotype due to a lack of cell elongation. Dwarfism could be rescued by the application of brassinolide, suggesting that DWF4 plays a role in brassinosteroid (BR) biosynthesis. The DWF4 locus is defined by four mutant alleles. One of these is the result of a T-DNA insertion. Plant DNA flanking the insertion site was cloned and used as a probe to isolate the entire DWF4 gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450 monooxygenase with 43% identity to the putative Arabidopsis steroid hydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM. Sequence analysis of two other mutant alleles revealed deletions or a premature stop codon, confirming that DWF4 had been cloned. This sequence similarity suggests that DWF4 functions in specific hydroxylation steps during BR biosynthesis. In fact, feeding studies utilizing BR intermediates showed that only 22alpha-hydroxylated BRs rescued the dwf4 phenotype, confirming that DWF4 acts as a 22alpha-hydroxylase.     

BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain-derived neurotrophic factor (BDNF) under the alpha-myosin heavy-chain promoter, as well as in BDNF knockout (-/-) mice. Compared with wild-type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase-immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase-or neuropeptide Y-IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (-/-) mice contained significantly fewer tyrosine hydroxylase-IR dermal nerve fibres than wild-type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF-dependence in vivo.     

Age-related effects of nerve growth factor on the morphology of embryonic sensory neurons in vitro

Studies of neonatal and adult mammals have shown that neuronal morphology is regulated in part by the availability of target-derived neurotrophic factor. To test whether the same is true for embryonic neurons, which are dependent on target-derived neurotrophic factors for survival, we grew neural crest-derived sensory neurons from the trigeminal ganglion of avian embryos of different ages in vitro in different concentrations of nerve growth factor (NGF) and measured the number of branch points and total length of the resulting arborizations. Although the size and complexity of arborizations increased with embryonic age up to embryonic day (E)14, neuronal morphology for embryos younger than E14 was unaffected by the concentration of NGF in the culture medium. However, beginning at E14, the stage at which trigeminal neurons start to lose their absolute requirement for NGF for survival, the neurons had significantly more branch points and larger arborizations in higher concentrations of NGF. Thus, it appears that the extent of neurite outgrowth in young embryos is independent of neurotrophic factor concentration; each neuron that receives enough neurotrophic factor to survive elaborates approximately the same size arbor. As trigeminal neurons mature and become less dependent on neurotrophic factor for survival, they acquire the ability to respond to neurotrophic factor with increased neurite growth and branching, as in neonates and adults.     

Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons

In the present study the ability of nerve growth factor (NGF) to facilitate the recovery of peptidergic primary sensory C-fibers after an acute capsaicin treatment (50 mg/kg s.c.) was investigated in adult rats. NGF (4 micrograms 1/day for 3 days) was injected into the plantar of one hind paw starting 24 h after the capsaicin treatment. Without NGF, there was a significant reduction of calcitonin gene-related peptide (CGRP) and substance P content of the paw skin and the sciatic nerve. CGRP and substance P levels were completely replenished in the NGF-treated paw skin and in the innervating sciatic nerve they even increased over control levels as determined 40 h after the last injection of NGF. CGRP levels also recovered in the contralateral paw and sciatic nerve, but no recovery was observed in other tissues such as the front paw, the auricle, or the urinary bladder. Mustard oil-induced neurogenic plasma extravasation, taken as a functional parameter for peptidergic primary sensory C-fibers, was significantly decreased after the capsaicin treatment and showed a complete recovery by NGF in the injected paw as well as in the contralateral paw skin. These results show that NGF not only was able to reverse the decrease of transmitter content caused by capsaicin but also restored the peripheral function of primary afferent neurons.     

Over-expression of NGF in skin causes formation of novel sympathetic projections to trkA-positive sensory neurons

Previous studies have shown that transgenic mice over-expressing NGF in the skin have novel sympathetic 'basket-like' projections to sensory neurons similar to that seen in models of chronic pain. Since only a subset of sensory neurons in the NGF-transgenic mice received the sympathetic projections, we hypothesized that sympathetic sprouting was targeted to those neurons affected by increased levels of NGF. To test this, double-label immunohistochemistry for the NGF receptor (trkA) and sympathetic baskets was performed. Thirty-nine percent of all neurons in transgenic trigeminal ganglia were trkA-positive. Moreover, of the population of sensory neurons that received sympathetic input, 84% were trkA-positive. These results indicate that retrogradely transported NGF can induce and direct growth of sympathetic axons in vivo.     

Overexpression of transforming growth factor beta1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor beta1 (TGF-beta1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-beta1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-beta1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-beta1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-beta1 in vascular disease.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.     

Locally administered vascular endothelial growth factor cDNA increases survival of ischemic experimental skin flaps

Both end-inspiratory (EIO) and end-expiratory (EEO) airway occlusions are used to calculate the strength of the Hering-Breuer inflation reflex (HBIR) in infants. However, the influence of the timing of such occlusions is unknown, as is the extent to which changes in volume within and above the tidal range affect this reflex. The purpose of this study was to compare both techniques and to evaluate the volume dependency of the HBIR in healthy, sleeping infants up to 1 yr of age. The strength of the HBIR was expressed as the ratio of expiratory or inspiratory time during EIO or EEO, respectively, to that recorded during spontaneous breathing, i.e., as the "inhibitory ratio" (IR). Paired measurements of the EIO and EEO in 26 naturally sleeping newborn and 15 lightly sedated infants at approximately 1 yr showed no statistically significant differences in the IR according to technique: mean (95% CI) of the difference (EIO - EEO) being -0.02 (-0.17, 0.13) during the first week of life and 0.04 (-0.14, 0.22) at 1 yr. During tidal breathing, a volume threshold of approximately 4 ml/kg was required to evoke the HBIR. Marked volume and age dependency were observed. In newborn infants, occlusions at approximately 10 ml/kg during sighs always resulted in an IR > 4, whereas a similar response was only evoked at 25 ml/kg in older infants. Age-related changes in the volume threshold may reflect maturational changes in the control of breathing and respiratory mechanics throughout the first year of life.     

Development of sensory innervation in dentin

We have labeled dental nerves of 3-week- to 1-year-old rats by axonal transport of radioactive protein in order to detect nerves in developing dentin by autoradiography. We found that, in addition to nerve growth, two processes determine adult dentinal nerve location: (1) enclosure of nerves within circumpulpal dentinal tubules during the last few weeks of dentinogenesis, beginning at the tip of the pulp horn and spreading to include most coronal dentin; and (2) gradual loss of the nerves near the tip of the cusp because of dentinal attrition and replacement by noninnervated, reparative dentin. Several days before a molar erupts, nerves at the tip of the cusp have already begun to be enclosed by dentin; 2-3 weeks later, when dentinogenesis at the cusp tip slows down, most of the innervation for that region has been established, nerves extend up to 160 micron into dentin, and the molar has reached the stage of functional occlusion. Soon after initial occlusion, the process of dentinal attrition and replacement by noninnervated reparative dentin begins; however, simultaneous dentinogenesis in more apical regions produces new innervated dentin. We studied nerve position in relation to dentin growth lines and found that there is a long-term association of nerve endings with specific sites in the dentinal tubules. The similarities between the development of dentinal innervation in rat molars and in human permanent teeth are discussed; it is found that as teeth mature and become more sensitive to painful stimuli, the density of dentinal nerves increases.     

Age-related changes in sympathetic modulation of sensory nerve activity in rat skin

OBJECTIVES: Sensory nerves play an important role in mediating neurogenic inflammation and subsequent tissue healing. A decrease in sensory nerve function with increasing age has been reported to correlate with poor tissue healing. Sympathetic nerves are known to modulate sensory nerve function, and changes in this modulation could also have important implications with ageing. The aims of this study were to examine the effect of different frequency electrical stimulation (ES) on the microvascular responses obtained to sensory nerve activation in young, aged and capsaicin-pretreated rats and modulation of these responses by sympathetic efferents. METHODS: Using laser Doppler flowmetry, vascular responses to antidromic ES of the sciatic nerve were monitored in the base of vacuum-induced blisters in the hind footpad. The non-selective alpha-adrenoceptor antagonist phentolamine (3 mg/kg, i.v.) was administered 20 min prior to ES. RESULTS: At high frequency ES (20V, 2ms, 15Hz for 1 min), the vascular response in old rats was significantly reduced (46 percent decrease, p < 0.05) compared to young control. At low frequency ES (20 V, 2 ms, 5 Hz for 1 min) however, older rats produced similar vascular responses to the young. Capsaicin-pretreated rats showed significantly reduced vascular responses to both high and low frequency ES, regardless of age. Pretreatment with phentolamine significantly increased the microvascular response in young rats at high (87 percent) and low (36 percent) frequency ES. In contrast, phentolamine significantly increased the ES-induced response in old rats at high frequency only (147 percent increase). CONCLUSIONS: The results suggest that the aged sensory nerve responds preferentially to low frequency ES and that sympathetic efferents exert an inhibitory modulatory effect on the vascular response evoked by sensory nerve stimulation. There are age-related changes in sympathetic modulation of sensory nerve-mediated responses which is dependent on stimulation frequency.     

p75-deficient sensory axons are immunoreactive for the glycoprotein L1 in mice overexpressing nerve growth factor

Nerve growth factor (NGF) regulates the expression of the glycoprotein L1 among neural cell populations. The purpose of this investigation was to determine whether NGF equally affects the immunolocalization of L1 on both sympathetic and sensory axons, and whether the functional expression of the p75 neurotrophin receptor (p75NTR) is required for the immunodetection of this glycoprotein on peripheral axons. Two lines of transgenic mice overexpressing NGF among glial cells were used in this study: (1) one line of mice possessing two normal alleles for p75NTR, and (2) another line of mice possessing two mutated alleles for p75NTR. In both types of animals, sensory axons stained immunohistochemically for calcitonin gene-related peptide and sympathetic axons stained immunohistochemically for tyrosine hydroxylase invaded the deep white matter portions of the cerebellum (a central structure containing high levels of transgene expression and synthesis); the cerebella of wild type (C57Bl/6) and p75NTR-deficient mice lacked these sensory and sympathetic fibers. Both lines of transgenic animals also possessed a dense plexus of L1-immunoreactive axons in their cerebella; the spatial distribution of these L1-immunostained axons paralleled that seen for the sensory and sympathetic axons. A unilateral removal of the superior cervical ganglion in both lines of transgenic animals caused a complete reduction of tyrosine hydroxylase-immunopositive axons in the cerebellum but did not affect the density of L1-immunopositive axons. From these in vivo data, we conclude that collateral branches of sensory axons which invade a NGF-rich target area display L1 immunoreactivity, and that such immunodetection does not require the functional expression of p75NTR.     

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers

Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26-41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers.     

Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for nonendothelial targets for VEGF

Vascular endothelial growth factor (VEGF) is a key regulator of endothelial growth and permeability. However, VEGF may also target nonendothelial cells, as VEGF receptors and responsiveness have been detected for example in monocytes, and high concentrations of VEGF have been reported in human semen. In this work we present evidence that overexpression of VEGF in the testis and epididymis of transgenic mice under the mouse mammary tumor virus (MMTV) LTR promoter causes infertility. The testes of the transgenic mice exhibited spermatogenic arrest and increased capillary density. The ductus epididymidis was dilated, containing areas of epithelial hyperplasia. The number of subepithelial capillaries in the epididymis was also increased and these vessels were highly permeable as judged by the detection of extravasated fibrinogen products. Intriguingly, the expression of VEGF receptor-1 (VEGFR-1) was detected in certain spermatogenic cells in addition to vascular endothelium, and both VEGFR-1 and VEGFR-2 were also found in the Leydig cells of the testis. The infertility of the MMTV-VEGF male mice could thus result from VEGF acting on both endothelial and nonendothelial cells of the male genital tract. Taken together, these findings suggest that the VEGF transgene has nonendothelial target cells in the testis and that VEGF may regulate male fertility.     

Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.     

The protective effect of the nerve growth factor in exposing a nerve tissue culture to diphtheria toxin

Diphtheria toxin (1.10(-1)-1.10(-6) Lf/ml) was found to inhibit neurite extension in chick embryo dorsal root ganglia in vitro. If the nerve growth factor (60 ng/ml) was added with toxin in culture media the diphtheria toxin effect was decreased and the neurite outgrowth was compared with control. Protective effect of nerve growth factor by influence of diphtheria toxin may be used in new principles of diphtheria treatment.     

Antisense oligonucleotide targeting the transforming growth factor beta1 increases expression of specific genes and functions of Leydig cells

OBJECTIVE: Effects of pacing-induced tachycardia on left ventricular function have been studied extensively. However, little attention has been focused on aortic elastic properties during heart rate increments. The aim was to determine the effects of right ventricular pacing on the aortic elastic properties. METHODS: We studied 14 normal subjects (baseline blood pressure, 129/84 +/- 10/6 mmHg; aortic diameter, 23.5/21.3 +/- 2.4/1.9 mm) at rest, during rapid right ventricular pacing (at five stepwise heart rate increases of 20 bpm every 2 min) and after 5 min recovery. Shifts as well as changes in the slope and the stiffness constant of the pressure diameter (p-d) relation, derived from simultaneous tip-micromanometer aortic pressure recordings and high-fidelity ultrasonic intravascular aortic diameter recordings, were used as indices of aortic stiffness. Wave reflection was also studied. RESULTS: Aortic pulse pressure and strain significantly decreased after pacing-induced tachycardia (p < 0.0001 and < 0.05, respectively). During pacing, the slope of the linear p-d relation as well as the stiffness constant were decreased, followed by increases at recovery (p < 0.0001). The augmentation index and the aortoventricular coupling ratio were significantly decreased (p < 0.0001). CONCLUSIONS: Pacing-induced increases in pulse frequency may result in improved aortic distensibility and aortoventricular coupling.