Zhiheng He

Progranulin (granulin-epithelin precursor, PC-cell-derived growth factor, acrogranin) mediates tissue repair and tumorigenesis.

Progranulin (Pgrn) is a pluripotent secreted growth factor that mediates cell cycle progression and cell motility. It activates the extracellular regulated kinases and phosphatidyl inositol-3 kinase signal cascades, among others, and increases expression of cyclins D and B. Structurally, it belongs to none of the well-established growth factor families. It regulates developmental events as diverse as the onset of cavitation in the preimplantation embryo and male-specific brain differentiation. During wound repair it promotes granulation and neovascularization. It regulates inflammation through a tripartite loop with secretory leukocyte protease inhibitor (SLPI) which protects pgrn from proteolysis, and elastase, which digests it to smaller peptides. Intact pgrn is anti-inflammatory through the inhibition of some of the actions of tumor necrosis factor, while the proteolytic peptides may stimulate the production of proinflammatory cytokines such as interleukin 8. Pgrn is highly expressed in aggressive cancer cell lines and clinical specimens including breast, ovarian, and renal cancers as well as gliomas. In experimental systems it confers an aggressive phenotype on poorly tumorigenic epithelial cancer cells. The malignancy of highly tumorigenic progranulin-expressing cell lines depends on the expression level of the pgrn gene since attenuating pgrn mRNA levels in pgrn-responsive cells greatly inhibits tumor progression. Given its actions in wound repair and tumorigenesis pgrn may prove a useful clinical target, both for prognosis and for therapy.

Cellular localization of gene expression for progranulin.

Granulins, also called epithelins, are 6-kD peptides with growth modulatory effects on a variety of cells. The granulin/epithelin precursor supports tumorigenesis in appropriate cell models and is the only growth factor able to overcome the cell cycle block that occurs in murine fibroblasts after deletion of a functional IGF-1 receptor. However, little is known of the role of granulin/epithelin gene products in vivo. To understand the physiological role of granulins it is essential to know the cell types and conditions in which it is expressed. We examined granulin/epithelin gene expression in adult rodents by in situ hybridization. The granulin/epithelin precursor is constitutively expressed in a number of epithelia, particularly in the skin, GI tract, and reproductive system. Other epithelia express the gene less strongly. Progranulin is expressed in immune cells in vivo and in specific neurons in the brain, including Purkinje cells, pyramidal cells of the hippocampus, and some cerebral cortical neurons. Little expression was detected in muscle cell, connective tissue, or endothelium. Cumulatively, these results define the basal gene expression of a new growth factor system and suggest that the progranulin/epithelin gene is multifunctional, with important constitutive roles in epithelial homeostasis, reproductive, immunological, and neuronal function.

Regulation of Vascular Endothelial Growth Factor Expression and Vascularization in the Myocardium by Insulin Receptor and PI3K/Akt Pathways in Insulin Resistance and Ischemia

Objective—This study characterized the role of insulin receptors and resistance on vascular endothelial growth factor (VEGF) expression and myocardial vascularization in physiological conditions and after ischemia. Methods and Results—Cardiac microvascular density was reduced by 30% in insulin-resistant Zucker fatty rats versus lean controls. This was associated with a parallel 40% inhibition of insulin-stimulated activation of both Akt and VEGF expression in the myocardium and cardiomyocytes. In contrast, the activation of Erk1/2 by insulin remained unchanged. In cultured cardiomyocytes, insulin or insulin-like growth factor (IGF)-1 increased VEGF mRNA and protein expression by 2-fold. Inhibition of PI3K/Akt, especially Akt2-mediated cascades but not the Ras/MEK/Erk pathway, using chemical inhibitors, dominant negative adenoviral constructs, or siRNA approaches suppressed VEGF mRNA expression by insulin. Ventricular tissues from muscle insulin receptor knockout (MIRKO) mice, which lack insulin receptors in the myocardium, have significant reductions in insulin but not IGF-1 signaling, VEGF expression, and vascular density before and after ischemia versus controls. Conclusions—Insulin regulates VEGF gene expression and vascularization in the myocardium specifically via insulin receptors and the activation of PI3K/Akt pathway. Selective inhibition of this pathway may lead to the decreases in VEGF expression and capillary density in the myocardium of patients with insulin resistance.

Differential Regulation of Angiotensin II-induced Expression of Connective Tissue Growth Factor by Protein Kinase C Isoforms in the Myocardium

Protein kinase C (PKC) and angiotensin II (AngII) can regulate cardiac function in pathological conditions such as in diabetes or ischemic heart disease. We have reported that expression of connective tissue growth factor (CTGF) is increased in the myocardium of diabetic mice. Now we showed that the increase in CTGF expression in cardiac tissues of streptozotocin-induced diabetic rats was reversed by captopril and islet cell transplantation. Infusion of AngII in rats increased CTGF mRNA expression by 15-fold, which was completely inhibited by co-infusion with AT1 receptor antagonist, candesartan. Similarly, incubation of cultured cardiomyocytes with AngII increased CTGF mRNA expression by 2-fold, which was blocked by candesartan and a general PKC inhibitor, GF109203X. The role of PKC isoform-dependent action was further studied using adenoviral vector-mediated gene transfer of dominant negative (dn) PKC or wild type PKC isoforms. Expression of dnPKCα, -ϵ, and -ζ isoforms suppressed AngII-induced CTGF expression in cardiomyocytes. In contrast, expression of dominant negative PKCδ significantly increased AngII-induced CTGF expression, whereas expression of wild type PKCδ inhibited this induction. This inhibitory effect was further confirmed in the myocardium of transgenic mice with cardiomyocyte-specific overexpression of PKCδ (δTg mice). Thus, AngII can regulate CTGF expression in cardiomyocytes through a PKC activation-mediated pathway in an isoform-selective manner both in physiological and diabetic states and may contribute to the development of cardiac fibrosis in diabetic cardiomyopathy.

Molecular Targets of Diabetic Cardiovascular Complications

Both the macro- and microvascular complications adversely affect the life quality of patients with diabetes and have been the leading cause of mortality and morbidity in this population. With the advancement of technologies in biomedical research, we have gained a great deal of understanding of the mechanisms underlying these complications. While euglycemic control still remains the best strategy, it is often difficult to maintain at a level that can completely prevent the vascular complications. Therefore, it is necessary to use the processes leading to vascular dysfunction as a framework for designing novel molecular therapeutic targets. Several of the mechanisms by which diabetes induces vascular complications include increased flux through the polyol pathway, increased oxidative stress, activation of protein kinase C (PKC), vascular inflammation, and abnormal expression and actions of cytokines in the vasculature. Many of the therapies that target these pathways have proven successful in experimental models of diabetic complications. However, clinical studies using these treatments have mainly yielded inconclusive results. The pathogenesis of diabetic vascular complications and results from animal studies and key clinical studies are reviewed here.

Effects of Insulin Replacements, Inhibitors of Angiotensin, and PKCβ's Actions to Normalize Cardiac Gene Expression and Fuel Metabolism in Diabetic Rats

High-density oligonucleotide arrays were used to compare gene expression of rat hearts from control, untreated diabetic, and diabetic groups treated with islet cell transplantation (ICT), protein kinase C (PKC)β inhibitor ruboxistaurin, or ACE inhibitor captopril. Among the 376 genes that were differentially expressed between untreated diabetic and control hearts included key metabolic enzymes that account for the decreased glucose and increased free fatty acid utilization in the diabetic heart. ICT or insulin replacements reversed these gene changes with normalization of hyperglycemia, dyslipidemia, and cardiac PKC activation in diabetic rats. Surprisingly, both ruboxistaurin and ACE inhibitors improved the metabolic gene profile (confirmed by real-time RT-PCR and protein analysis) and ameliorated PKC activity in diabetic hearts without altering circulating metabolites. Functional assessments using Langendorff preparations and 13C nuclear magnetic resonance spectroscopy showed a 36% decrease in glucose utilization and an impairment in diastolic function in diabetic rat hearts, which were normalized by all three treatments. In cardiomyocytes, PKC inhibition attenuated fatty acid–induced increases in the metabolic genes PDK4 and UCP3 and also prevented fatty acid–mediated inhibition of basal and insulin-stimulated glucose oxidation. Thus, PKCβ or ACE inhibitors may ameliorate cardiac metabolism and function in diabetes partly by normalization of fuel metabolic gene expression directly in the myocardium.

Selective Regulation of Heme Oxygenase-1 Expression and Function by Insulin through IRS1/Phosphoinositide 3-Kinase/Akt-2 Pathway

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H2O2-induced NF-κB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Protein Kinase Cβ Isoform Inhibitors A New Treatment for Diabetic Cardiovascular Diseases

The center of the pandemic of diabetes is the life-threatening cardiovascular complications that can be categorized into macrovasculopathy, microvasculopathy, and diabetic cardiomyopathy. Macrovasculopathy affects large vessels and manifests as atherosclerosis and the subsequent coronary artery disease, peripheral artery disease, and cerebrovascular disease. Atherosclerosis and its related complications are responsible for most of the mortality in diabetic patients.1 Indeed, diabetes has been firmly established as an independent risk factor for atherosclerosis. This risk seems to precede the onset of type 2 diabetes but begins with hyperglycemia in patients with type 1 diabetes.2 Multiple factors in diabetes—including insulin resistance, dyslipidemia, elevated free fatty acid, and hypertension—increase the risk. Many clinical surveys, such as the Framingham Study, have shown that the incidence of carotid artery disease is increased 2- to 4-fold in patients with diabetes or insulin resistance. When intima-media thickness is used as the indicator for the severity of atherosclerosis, both type 13,4 diabetes and type 25 diabetes are associated with more advanced atherosclerosis when compared with age- and sex-matched controls. This association could also be related to hyperglycemia because intensive euglycemic control has been shown to reduce the progression of intima-media thickness in the Epidemiology of Diabetes Interventions and Complications study, which involves 1229 patients.6 These clinical data suggest that hyperglycemia itself, in addition to the aforementioned established risk factors such as dyslipidemia, hypertension, insulin resistance, and oxidative stress, might have an independent role in the acceleration of atherosclerosis. See p 91 Diabetes or insulin resistance clearly alters the biology of the multiple cellular components that participate in atherosclerosis, including endothelial cells, monocytes/macrophages, lymphocytes, and vascular smooth muscle cells. In endothelial cells, both the expression and the activity of the endothelial cell nitric oxide synthase are decreased, leading to the reduction of antiatherogenic nitric oxide (NO) …

Phosphoinositide 3-kinase Akt signaling pathway interacts with protein kinase Cβ2 in the regulation of physiologic developmental hypertrophy and heart function

The phosphoinositide 3-kinase (PI3-kinase)-protein kinase B (Akt) signaling pathway is essential in the induction of physiological cardiac hypertrophy. In contrast, protein kinase C beta2 (PKCbeta2) is implicated in the development of pathological cardiac hypertrophy and heart failure. Thus far, no clear association has been demonstrated between these two pathways. In this study, we examined the potential interaction between the PI3-kinase and PKCbeta2 pathways by crossing transgenic mice with cardiac specific expression of PKCbeta2, constitutively active (ca) PI3-kinase, and dominant-negative (dn) PI3-kinase. In caPI3-kinase/PKCbeta2 and dnPI3-kinase/PKCbeta2 double-transgenic mice, the heart weight-to-body weight ratios and cardiomyocyte sizes were similar to those observed in caPI3-kinase and dnPI3-kinase transgenic mice, respectively, suggesting that the regulation of physiological developmental hypertrophy via modulation of cardiomyocyte size proceeds through the PI3-kinase pathway. In addition, we observed that caPI3-kinase/PKCbeta2 mice showed improved cardiac function while the function of dnPI3-kinase/PKCbeta2 mice was similar to that of the PKCbeta2 group. PKCbeta2 protein levels in both dnPI3-kinase/PKCbeta2 and PKCbeta2 mice were significantly upregulated. Interestingly, however, PKCbeta2 protein expression was significantly attenuated in caPI3-kinase/PKCbeta2 mice. PI3-kinase activity measured by Akt phosphorylation was not affected by PKCbeta2 overexpression. These data suggest a potential interaction between these two pathways in the heart, where PI3-kinase is predominantly responsible for the regulation of physiological developmental hypertrophy and may act as an upstream modulator of PKCbeta2 with the potential for rescuing the pathological cardiac dysfunction induced by overexpression of PKCbeta.

Modulating Notch signaling to enhance neovascularization and reperfusion in diabetic mice.

Diabetes can diminish the responsiveness to angiogenic factors (e.g., VEGF) important for wound healing and the treatment of ischemic diseases, and this study investigated the hypothesis that this effect can be reversed by altering Notch signaling. Aortic endothelial cells (ECs) isolated from diabetic mice demonstrated reduced sprouting capability in vitro, but adding a Notch inhibitor (DAPT) led to cell-density and VEGF-dose dependent enhancement of proliferation, migration and sprouting, in both 2-D and 3-D cultures, as compared to VEGF alone. The in vivo effects of VEGF and DAPT were tested in the ischemic hind limbs of diabetic mice. Combining VEGF and DAPT delivery resulted in increased blood vessel density (∼150%) and improved tissue perfusion (∼160%), as compared to VEGF alone. To examine if DAPT would interfere with vessel maturation, DAPT was also delivered with a combination of VEGF and platelet derived growth factor (PDGF). DAPT and PDGF did not interfere with the effects of the other, and highly functional and mature networks of vessels could be formed with appropriate delivery. In summary, modulating Notch signaling enhances neovascularization and perfusion recovery in diabetic mice suffering from ischemia, suggesting this approach could have utility for human diabetics.