The chemokine (C-X-C motif) receptor 4 inhibitor AMD3100 accelerates blood flow restoration in diabetic mice.

AIMS/HYPOTHESIS: Bone marrow cell mobilisation potently induces vascular growth in ischaemic tissue, possibly by mobilising endothelial cell progenitors. Thus, mobilising agents might not be therapeutic when endothelial cell progenitors are dysfunctional, as in diabetes mellitus. Local injection of autologous endothelial cell progenitors also stimulates vascular growth in ischaemic tissue, but endothelial cell progenitors from people with multiple cardiovascular risk factors and from obese diabetic mice are marginally therapeutic or inhibitory. We sought to identify possible strategies to improve vascularisation in patients with diabetes mellitus by determining if (1) mobilisation accelerates neovascularisation in diabetic animals, and (2) mobilised cells from a non-diabetic source accelerate vascularisation in diabetic animals. METHODS: We tested whether systemic administration of the chemokine (C-X-C motif) receptor 4 inhibitor AMD3100 or local injection of human CD34(+) circulating cells mobilised by AMD3100 could speed or enhance blood flow restoration in ischaemic limbs of diabetic mice. The small-molecule-mobilising drug AMD3100 was selected because mobilisation and apheresis can be done on the same day. RESULTS: Systemic administration of AMD3100 and local injection of cells mobilised by AMD3100 greatly accelerated the restoration of blood flow to ischaemic limbs of diabetic mice. CD34(+) cells mobilised by AMD3100 appeared to be more potent growth stimulators than their unmobilised counterparts. CONCLUSIONS/INTERPRETATION: Unlike other mobilising agents requiring multi-day mobilisation, AMD3100 enables mobilised donors to undergo mobilisation and apheresis on the same day. The combination of excellent therapeutic benefits as well as ease of use indicates that AMD3100 could be a powerful tool to ameliorate tissue ischaemia in the diabetic environment.

CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds.

Diabetes is characterized by poor circulation and impaired angiogenesis, which appear to contribute to the frequent skin lesions and poor wound healing common in diabetic patients. Therapies to improve circulation commonly improve wound healing in diabetic patients. Administration of circulating CD34+ cells, cells that can function as endothelial cell progenitors, accelerates blood flow restoration to ischemic limbs of diabetic mice. We have investigated the potential of these cells to accelerate revascularization and healing in full-thickness skin wounds of hypoinsulinemic (streptozotocin-treated) diabetic mice. Wounds were injected with human CD34+ or CD34- peripheral blood mononuclear cells or no cells, and analyzed for vascularity and healing at various times thereafter. Treatment with CD34+ enriched cells decreased wound size by 4 days after treatment, accelerated epidermal healing, and rapidly and dramatically accelerated revascularization of the wounds compared to controls. Initially increased vascularization was mediated principally by an increase in vessel diameter, but later, both an increase in vascular size and number were observed. These findings indicate that blood-derived progenitors may have therapeutic potential in the treatment of skin lesions in the setting of diabetes, and give insights into how bone marrow cells exert their effects on neovascularization.

CD34- blood-derived human endothelial cell progenitors.

A subset of adult peripheral blood leukocytes functions as endothelial cell progenitors called angioblasts. They can incorporate into the vasculature in animal models of neovascularization and accelerate the restoration of blood flow to mouse ischemic limbs. Earlier reports suggested that CD34-expressing (CD34+) but not CD34+ cell-depleted (CD34-) leukocytes can differentiate into endothelial cells (EC) in vitro and in vivo. Recent findings suggest that CD14+ cells, which are typically CD34-, also have angioblast-like properties in vitro. To determine the identity of angioblasts, the potential of CD34+, CD34-, CD34- CD14+, and CD34- CD14- cells to produce EC was compared. We show that a subset of monocyte (CD34- CD14+)-enriched cells can take on an EC-like phenotype in culture, but that the EC-like cells also express dendritic cell antigens. These findings suggest that monocytes differentiate into macrophages, dendritic cells, or EC depending on environmental cues. The data also demonstrate that angioblasts are more abundant in the blood than previously thought. Finally, we demonstrate that CD34- and CD34- CD14+ cells incorporate into the endothelium of blood vessels in mouse ischemic limbs. However, incorporation of these cells requires co-injection with CD34+ cells, indicating that leukocyte-leukocyte interactions may play a critical role in governing angioblast behavior in vivo.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry.

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice.

Endothelial cell progenitors, angioblasts, have been detected in the peripheral blood of adult humans, mice, and rabbits. These cells have been shown to incorporate into the endothelium of newly forming blood vessels in pathological and nonpathological conditions. Here we investigated the possibility that the CD34-expressing leukocytes (CD34(+) cells) that appear to be enriched for angioblasts could be used to accelerate the rate of blood-flow restoration in nondiabetic and diabetic mice undergoing neovascularization due to hindlimb ischemia. CD34(+) cells did not accelerate the restoration of flow in nondiabetic mice, but dramatically increased it in diabetic mice. Furthermore, CD34(+) cells derived from type 1 diabetics produced fewer differentiated endothelial cells in culture than did their type 2 diabetic- or nondiabetic-derived counterparts. In vitro experiments suggest that hyperglycemia per se does not alter the ability of angioblasts to differentiate or of angioblast-derived endothelial cells to proliferate. In contrast, hyperinsulinemia may enhance angioblast differentiation but impair angioblast-derived endothelial cell survival or proliferation. Our findings suggest that CD34(+) cells may be a useful tool for therapeutic angiogenesis in diabetics.

Angiogenesis: new insights and therapeutic potential.

Angiogenesis, the formation of vessels from pre-existing vessels, is of critical importance not only during normal growth, but also in pathological situations. In the latter, some diseases are enhanced by excessive vascular growth (e.g., tumors), whereas in others inadequate vascular growth contributes to morbidity and mortality (e. g., ischemic heart disease). Our current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell-cell interactions, various intracellular signaling pathways, and the appropriate extracellular microenvironment. The literature regarding angiogenesis has increased exponentially during the last decade. Progress in this area is largely a consequence of advances in our understanding of angiogenic growth factor and cytokine function, in part due to the determination of their complete amino acid sequences and cloning of their genes. Other factors also play key roles in angiogenesis, including the extracellular matrix, adhesion molecules and their inhibitors, and metabolic and mechanical factors. The potential for developing therapeutic protocols has been enhanced by data from both in vitro and in vivo studies and has provided the rationale for clinic trials. Angiogenic therapy strategies include inhibition of aberrant angiogenesis, as seen in tumors or diabetes, as well as stimulation of angiogenesis in conditions of ischemia, such as ischemic heart or peripheral vascular disease. Anat Rec (New Anat) 261:126-135, 2000.

Expression of the dominant-negative regulator Id4 is induced during adipocyte differentiation.

Expression of the Id4 gene was investigated during differentiation of 3T3-L1 preadipocytes into mature adipocytes. Id4 is a member of a family encoding non-DNA binding helix-loop-helix proteins proposed to inhibit the activity of basic HLH (bHLH) proteins important in many developmental processes. We show here that Id4 expression is low in confluent preadipocytes and rapidly induced by treatment with the combination of hormones which causes differentiation into mature adipocytes. Id4 expression is also induced by treatment with individual hormones, especially dexamathasone. Id4 mRNA can be detected in mouse and human adipose tissue. Genes encoding E-proteins (bHLH proteins known to interact with and be regulated by Id proteins) are expressed and regulated during differentiation in 3T3-L1 cells. These data suggest that the Id4 transcriptional regulator is playing a role in adipose cell differentiation and suggest that DNA-binding HLH proteins may also be important in regulation of differentiation of these cells.

Differential biological activities of mammalian Id proteins in muscle cells.

Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identified in vitro. Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.

Altered development of spinal cord in the mouse mutant (Patch) lacking the PDGF receptor alpha-subunit gene.

The platelet-derived growth factor receptor alpha subunit (PDGFR alpha) is expressed by glial precursors, glial cells, and some peripheral neurons during normal rodent development. Its ligands are expressed ubiquitously in neurons, including sensory and motor neurons. Thus, neuronally secreted PDGF-A may play a paracrine role in the development of both glial cells and peripheral neurons. The Patch (Ph) mutation, which is a deletion of the PDGFR alpha, is a homozygous embryonic lethal mutation in the mouse. Previously, several developmental abnormalities, including deficiencies in connective tissues in many organs, aberrant neural crest cell migration, and defects in non-neuronal derivatives of crest cells, have been shown to be associated with the Patch mutation. However, whether and the extent to which motor and sensory neurons are affected by the mutation are not known. Here, we have examined the survival and/or morphological differentiation of spinal motor and sensory (dorsal root ganglion) neurons during the period of naturally occurring cell death, i.e., between E14 and E18, in control and Ph/Ph mice. The results show a 65-70% decrease in motor and sensory neuron numbers in Ph/Ph mice, compared to controls, at all stages examined. Furthermore, motoneurons in Ph/Ph mice were significantly smaller than those in controls. Because of the bidirectional nature of neuron-glial cell interactions, these results suggest that PDGFR alpha plays an important role in glial cell development and, thus, indirectly in neuronal cell development or, alternatively, that PDGF and the PDGFR alpha are directly involved in peripheral neuron survival and development by an autocrine/paracrine mechanism.

Platelet-derived growth factor receptor alpha subunit deleted Patch mouse exhibits severe cardiovascular dysmorphogenesis.

Patch (Ph) mice, whose platelet-derived growth factor receptor alpha subunit (alpha PDGFR) gene has been deleted, have been used to elucidate requirements for alpha PDGFR for normal murine development. In this report we evaluate the role of alpha PDGFR in cardiovascular development by using in situ hybridization to follow the changing pattern of alpha PDGFR expression in cardiovascular tissues after embryonic day 13, and comparing this pattern with the pattern of cardiovascular defects observed in homozygous Ph mutants. Both mesodermally derived and neural crest-derived components of the cardiovascular system are severely dysmorphic in Ph/Ph embryos and those structures most severely affected are those that normally express alpha PDGFR mRNA at the highest levels and for the longest duration. Ph/Ph vessels appear to be lined with a normal endothelium, but contain a reduced number of smooth muscle cells and are fragile during processing for histology. The myocardium is thin, the heart is small and dysmorphic, the valves are malformed, and the interventricular and interatrial septa of the heart are defective. In the outflow tract, the spectrum of defects includes both persistent truncus arteriosus and double outlet right ventricle. This pattern of abnormalities is consistent with the hypothesis that deletion of alpha PDGFR results in a functional ablation of cranial neural crest cells, and that mesodermally derived components of the vascular system also require alpha PDGFR.

Nerve growth factor receptor expression in fetal, mature, and diseased human kidneys.

BACKGROUND: Nerve growth factor (NGF) has been proposed to be critical to normal renal development in rodents. However, little is known about expression of NGF or its receptors in human kidneys, or their potential function in development or disease. EXPERIMENTAL DESIGN: A previously characterized monoclonal antibody (NGFR 5) was utilized for immunohistochemical localization of the p75 NGF receptor (NGFR) in alcohol-fixed tissue sections of human fetal kidney (N = 27, 54 to 105 days gestation), normal mature kidney obtained from nephrectomies for neoplasia (N = 7), and renal biopsies (N = 54) with various glomerulopathies previously characterized for degree of mesangial alpha smooth muscle actin (alpha SM) expression. A second monoclonal antibody (NGFR2) was also utilized on fetal and normal kidney. Immunohistochemical localization of alpha SM and proliferating cell nuclear antigen expression was also performed. RESULTS: Glomerular expression of NGFR in the fetus is limited to the mesangium in later stages of glomerulogenesis; at these stages this expression is similar to that which has been previously reported for platelet-derived growth factor receptor-beta and alpha SM. There is focal, weak persistence of NGFR in normal adult glomeruli, similar to alpha SM. In renal biopsies, glomerular NGFR expression was upregulated in a variety of disease states, which frequently but not invariably correlated with alpha SM expression. Fetal and adult expression of NGFR is also prominent in periarterial connective tissue cells and nerve. Apparent de novo expression by many interstitial cells in normal and diseased adult kidneys is also present. CONCLUSIONS: These studies indicate: (a) NGF or other neurotrophins that bind NGFR may be important in human kidney development and glomerular response to injury; (b) NGFR is a marker of developing mesangial cells similar to alpha SM and platelet-derived growth factor receptor-beta; (c) enhanced expression of NGFR, like alpha SM, is a marker of mesangial cell injury or activation, and that their coordinate upregulation in some glomerular disease states appears to recapitulate a normal developmental state; (d) a population of NGFR and platelet-derived growth factor-beta expressing interstitial cells can be identified in normal kidneys, which suggests potential signaling mechanisms to recruit or activate these cells at sites of tubulointerstitial injury.

Distribution of the 75-kD low-affinity nerve growth factor receptor in the primate peripheral nervous system.

Disruption of the 75-kD low-affinity nerve growth factor (NGF) receptor (p75) has been shown to result in sensory and sympathetic nervous system deficits (Lee et al., 1992a,b). In order to establish precisely which subsets of neurons are capable of responding to neurotrophins (NTs) through the low-affinity NGF receptor, p75 was localized in the primate autonomic and somatic sensory nervous systems. In the autonomic system, cell bodies of some parasympathetic and enteric neurons expressed detectable levels of p75, whereas all sympathetic neurons expressed the protein. In the sensory system, some, but not all, cell bodies were labeled in cranial and spinal sensory ganglia and in the mesencephalic nucleus. Some peripheral and central projections of the sensory neurons were also labeled. Centrally, most of the labeled processes were found in regions containing primarily small unmyelinated fibers, including lamina II of Rexed and areas of the solitary tract and nucleus. Peripherally, labeled processes were associated with unmyelinated nerves and specialized structures such as taste buds and Meissner corpuscles, but not with myelinated processes. This study indicates that the subset of neurons in the autonomic nervous system likely to be capable of responding to neurotrophins is broader than generally thought, and that p75-expressing neurons tend to be clustered. Moreover, in the sensory nervous system p75 is expressed by most cell bodies, but expression in their projections is restricted both peripherally and centrally to unmyelinated processes and nerve terminals.

Regulation and role of PDGF receptor alpha-subunit expression during embryogenesis.

The platelet-derived growth factor receptor alpha-subunit (PDGFR alpha) is the form of the PDGF receptor that is required for binding of PDGF A-chain. Expression of PDGFR alpha within the early embryo is first detected as the mesoderm forms, and remains characteristic of many mesodermal derivatives during later development. By 9.5 days of development, embryos homozygous for the Patch mutation (a deletion of the PDGFR alpha) display obvious growth retardation and deficiencies in mesodermal structures, resulting in the death of more than half of these embryos. Mutant embryos that survive this first critical period are viable until a new set of defects become apparent in most connective tissues. For example, the skin is missing the dermis and connective tissue components are reduced in many organs. By this stage, expression of PDGFR alpha mRNA is also found in neural crest-derived mesenchyme, and late embryonic defects are associated with both mesodermal and neural crest derivatives. Except for the neural crest, the lens and choroid plexus, PDGFR alpha mRNA is not detected in ectodermal derivatives until late in development in the central nervous system. Expression is not detected in any embryonic endodermal derivative at any stage of development. These results demonstrate that PDGFR alpha is differentially expressed during development and that this expression is necessary for the development of specific tissues.

A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells.

The Patch (Ph) mutation in mice is a deletion of the gene encoding the platelet-derived growth factor receptor alpha subunit (PDGFR alpha). Patch is a recessive lethal recognized in heterozygotes by its effect on the pattern of neural crest-derived pigment cells, and in homozygous mutant embryos by visible defects in craniofacial structures. Since both pigment cells and craniofacial structures are derived from the neural crest, we have examined the differentiation of other crest cell-derived structures in Ph/Ph mutants to assess which crest cell populations are adversely affected by this mutation. Defects were found in many structures populated by non-neuronal derivatives of cranial crest cells including the thymus, the outflow tract of the heart, cornea, and teeth. In contrast, crest-derived neurons in both the head and trunk appeared normal. The expression pattern of PDGFR alpha mRNA was determined in normal embryos and was compared with the defects present in Ph/Ph embryos. PDGFR alpha mRNA was expressed at high levels in the non-neuronal derivatives of the cranial neural crest but was not detected in the crest cell neuronal derivatives. These results suggest that functional PDGF alpha is required for the normal development of many non-neuronal crest-derived structures but not for the development of crest-derived neuronal structures. Abnormal development of the non-neuronal crest cells in Ph/Ph embryos was also correlated with an increase in the diameter of the proteoglycan-containing granules within the crest cell migratory spaces. This change in matrix structure was observed both before and after crest cells had entered these spaces. Taken together, these observations suggest that functional PDGFR alpha can affect crest development both directly, by acting as a cell growth and/or survival stimulus for populations of non-neurogenic crest cells, and indirectly, by affecting the structure of the matrix environment through which such cells move.

Multiple functions for NGF receptor in developing, aging and injured rat teeth are suggested by epithelial, mesenchymal and neural immunoreactivity.

We have used immunocytochemistry to analyse expression of nerve growth factor receptor (NGFR) in developing, aging and injured molar teeth of rats. The patterns of NGFR immunoreactivity (IR) in developing epithelia and mesenchyme matched the location of NGFR mRNA assayed by in situ hybridization with a complementary S35-labeled RNA probe. The following categories of NGFR expression were found. (1) There was NGFR-IR in the dental lamina epithelium and in adjacent mesenchyme during early stages of third molar formation. (2) NGFR-IR nerve fibers were posterior and close to the bud epithelium. (3) During crown morphogenesis NGFR expression was prominent in internal enamel epithelium and preodontoblasts; it faded as preameloblasts elongated and as odontoblasts began to make predentin matrix; and it was weak or absent from outer enamel epithelium, the cervical loop, and differentiated ameloblasts and odontoblasts. (4) When NGFR-IR nerve fibers entered the molars late in the bell stage, they innervated the most mature peripheral pulp and dentin in an asymmetric pattern which correlated more with asymmetric enamel synthesis than with mesenchymal NGFR-IR distribution. (5) The mesenchymal pulp cells continued to have intense NGFR expression in adult teeth, especially near coronal tubular dentin. (6) The pulpal NGFR-IR decreased in very old rats or subjacent to reparative dentin (naturally occurring or experimentally induced). (7) During root formation, the preodontoblasts had NGFR-IR but most root mesenchymal cells and Hertwig's epithelial root sheath did not. This work suggests that there are important epithelial and mesenchymal targets of NGF regulation during molar morphogenesis that differ for crown and root development and that do not correlate with neural development. The continuing expression of NGFR-IR by pulpal mesenchymal cells in adult rats was most intense near coronal odontoblasts making tubular dentin; and it was lost during aging, or subjacent to sites of dentin injury that caused a phenotypic change in the odontoblast layer.

A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue.

The expression of human nerve growth factor (NGF) receptor in tumors and normal tissue was investigated with the use of a monoclonal antibody recently developed against that protein. This antibody, NGFR5, reacted strongly with 100% of 25 nerve sheath tumors. Eight of nine pheochromocytomas and three of three paragangliomas also had positive results, but the immunoreactivity was restricted to the sustentacular cell population. Within cells of melanocytic lineage, there was no immunostaining of melanocytes in normal epidermis, whereas 13 of 14 benign nevi had positive results, primarily involving spindled nevocytic structures within the dermis. NGF receptor was scarcely expressed in human melanoma; 9 of 19 melanomas had positive results, but immunoreactivity was generally restricted to rare cells within the larger tumor cell population. Among nonneurogenic mesenchymal tumors, results were generally negative: 0 of 5 chondrosarcomas, 0 of 6 malignant fibrous histiocytomas, 0 of 3 meningiomas, and 1 of 8 leiomyosarcomas were immunoreactive. Carcinomas were variable in immunoreactivity: 12 of 16 squamous cell carcinomas had positive results, whereas adenocarcinomas demonstrated focal, basal epithelial immunoreactivity and neuroendocrine tumors generally had negative results. Among normal tissues, in addition to expected neural immunostaining, NGFR 5 reacted positively with several nonneural cell types, including lymphoidal follicular dendritic cells, myoepithelial cells, vascular adventitia, and basal epithelium of oral mucosa and hair follicles. Antibodies to NGF receptor may play a role in the identification of benign and malignant soft tissue lesions.

Expression of NGF receptor in the developing and adult primate central nervous system.

Monoclonal antibodies against human NGF receptor have been used for immunocytochemical localization of NGF receptors in the CNS of macaques and baboons at various stages of development. In the adult, neurons in most brain regions are devoid of detectable NGF receptors. However, abundant NGF receptor immunoreactivity is present on a population of neurons in basal forebrain, which, on the basis of appearance and pattern of distribution, probably correspond, at least in part, to magnocellular cholinergic neurons of this region. NGF receptors were also associated with the vasculature in most brain regions. NGF receptor immunoreactivity is present on Mueller glia of neural retina. In macaque fetuses, approximately 1 month prenatally, retinal Mueller glia possess lower levels of receptor, while higher levels of receptor are present in the retinal nerve fiber layer. In fetal cerebellum, abundant receptor immunoreactivity is present on Purkinje cells, granule cells of the premigratory zone of the external granule layer, and neurons of the deep nuclei. Immunoreactivity decreases with subsequent development and is absent in the adult. In cerebellum, levels of NGF receptor assayed by affinity crosslinking to radioiodinated NGF, and levels of NGF receptor mRNA assayed by Northern blot analysis decrease dramatically during the last month of fetal life.