Connective tissue growth factor level is increased in patients with liver cirrhosis but is not associated with complications or extent of liver injury

Connective tissue growth factor (CTGF) is a profibrotic protein whose systemic levels are increased in liver cirrhosis. Here, association of CTGF with stages of liver injury and complications of cirrhotic liver disease has been analyzed in patients with different aetiologies of hepatic injury. CTGF is significantly increased in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 46 patients with liver cirrhosis compared to eight liver-healthy controls. In patients´ blood samples CTGF in HVS is about 6% higher than PVS levels indicating that CTGF produced in the liver is released to the circulation. CTGF is not associated with stages of liver cirrhosis defined by CHILD-PUGH or MELD score nor with secondary complications of portal hypertension (varices, ascites, spontaneous bacterial peritonitis). Transforming growth factor β (TGFβ) induces CTGF synthesis in hepatocytes and a positive association of systemic TGFβ1 and SVS and HVS CTGF is found. Three months after placing transjugular intrahepatic portosystemic shunt (TIPS) hepatic venous pressure gradient is reduced whereas CHILD-PUGH score, TGFβ1 and CTGF are not altered in serum of 15 patients. Current data show that the cirrhotic liver releases little CTGF but SVS, HVS and PVS CTGF levels are not associated with residual liver function and complications of cirrhosis.

Comparison of the gene expression profiles from normal and Fgfrl1 deficient mouse kidneys reveals downstream targets of Fgfrl1 signaling

Fgfrl1 (fibroblast growth factor receptor-like 1) is a transmembrane receptor that is essential for the development of the metanephric kidney. It is expressed in all nascent nephrogenic structures and in the ureteric bud. Fgfrl1 null mice fail to develop the metanephric kidneys. Mutant kidney rudiments show a dramatic reduction of ureteric branching and a lack of mesenchymal-to-epithelial transition. Here, we compared the expression profiles of wildtype and Fgfrl1 mutant kidneys to identify genes that act downstream of Fgfrl1 signaling during the early steps of nephron formation. We detected 56 differentially expressed transcripts with 2-fold or greater reduction, among them many genes involved in Fgf, Wnt, Bmp, Notch, and Six/Eya/Dach signaling. We validated the microarray data by qPCR and whole-mount in situ hybridization and showed the expression pattern of candidate genes in normal kidneys. Some of these genes might play an important role during early nephron formation. Our study should help to define the minimal set of genes that is required to form a functional nephron.

Vascular endothelial growth factor-A and aldosterone: relevance to normal pregnancy and preeclampsia

Aldosterone levels are markedly elevated during normal pregnancy but fall even though volume contracts when preeclampsia occurs. The level of aldosterone in either condition cannot be explained solely by the activity of the renin-angiotensin II system. In normal gestation, vascular endothelial growth factor (VEGF) is thought to maintain vascular health, but its role in adrenal hormone production is unknown. We hypothesized that the role of VEGF in the adrenal gland is to maintain vascular health and regulate aldosterone production. Here, we demonstrate that supernatant of endothelial cells grown in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with angiotensin II increased aldosterone production in adrenal cells. These data suggest that endothelial cell-dependent and independent activation of aldosterone is regulated by VEGF. In contrast to angiotensin II, VEGF did not upregulate the steroidogenic acute regulatory protein. Consistent with this observation, angiotensin II stimulated both aldosterone and cortisol synthesis from progesterone, whereas VEGF stimulated selectively aldosterone production. In rats, overexpression of soluble fms-like tyrosine kinase-1, an endogenous VEGF inhibitor, led to adrenocortical capillary rarefaction and fall in aldosterone concentrations that correlated inversely with soluble fms-like tyrosine kinase-1 levels. These findings may explain why aldosterone increases so markedly during normal gestation and why preeclampsia, a condition characterized by high soluble fms-like tyrosine kinase-1, is associated with inappropriately low aldosterone levels in spite of relatively lower plasma volumes.

PA21, a New Iron-Based Noncalcium Phosphate Binder, Prevents Vascular Calcification in Chronic Renal Failure Rats

Chronic renal failure (CRF) is associated with the development of secondary hyperparathyroidism and vascular calcifications. We evaluated the efficacy of PA21, a new iron-based noncalcium phosphate binder, in controlling phosphocalcic disorders and preventing vascular calcifications in uremic rats. Rats with adenine-diet-induced CRF were randomized to receive either PA21 0.5, 1.5, or 5% or CaCO3 3% in the diet for 4 weeks, and were compared with uremic and nonuremic control groups. After 4 weeks of phosphate binder treatment, serum calcium, creatinine, and body weight were similar between all CRF groups. Serum phosphorus was reduced with CaCO3 3% (2.06 mM; P ≤ 0.001), PA21 1.5% (2.29 mM; P < 0.05), and PA21 5% (2.21 mM; P ≤ 0.001) versus CRF controls (2.91 mM). Intact parathyroid hormone was strongly reduced in the PA21 5% and CaCO3 3% CRF groups to a similar extent (1138 and 1299 pg/ml, respectively) versus CRF controls (3261 pg/ml; both P ≤ 0.001). A lower serum fibroblast growth factor 23 concentration was observed in the PA21 5%, compared with CaCO3 3% and CRF, control groups. PA21 5% CRF rats had a lower vascular calcification score compared with CaCO3 3% CRF rats and CRF controls. In conclusion, PA21 was as effective as CaCO3 at controlling phosphocalcic disorders but superior in preventing the development of vascular calcifications in uremic rats. Thus, PA21 represents a possible alternative to calcium-based phosphate binders in CRF patients.

P450 oxidoreductase deficiency: a new form of congenital adrenal hyperplasia

PURPOSE OF REVIEW: P450 oxidoreductase deficiency--a newly described form of congenital adrenal hyperplasia--typically presents a steroid profile suggesting combined deficiencies of steroid 21-hydroxylase and 17alpha-hydroxylase/17,20-lyase activities. These and other enzymes require electron donation from P450 oxidoreductase. The clinical spectrum of P450 oxidoreductase deficiency ranges from severely affected children with ambiguous genitalia, adrenal insufficiency and the Antley-Bixler skeletal malformation syndrome to mildly affected individuals with polycystic ovary syndrome. We review current knowledge of P450 oxidoreductase deficiency and its broader implications. RECENT FINDINGS: Since the first report in 2004, at least 21 P450 oxidoreductase mutations have been reported in over 40 patients. The often subtle manifestations of P450 oxidoreductase deficiency suggest it may be relatively common. P450 oxidoreductase deficiency, with or without Antley-Bixler syndrome, is autosomal recessive, whereas Antley-Bixler syndrome without disordered steroidogenesis is caused by autosomal dominant fibroblast growth factor receptor 2 mutations. In-vitro assays of P450 oxidoreductase missense mutations based on P450 oxidoreductase-supported P450c17 activities provide excellent genotype/phenotype correlations. The causal connection between P450 oxidoreductase deficiency and disordered bone formation remains unclear. SUMMARY: P450 oxidoreductase mutations cause combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase. Individuals with an Antley-Bixler syndrome-like phenotype presenting with sexual ambiguity or other abnormalities in steroidogenesis should be analyzed for P450 oxidoreductase deficiency.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

Distinct roles of vascular endothelial growth factor-D in lymphangiogenesis and metastasis

In many human carcinomas, expression of the lymphangiogenic factor vascular endothelial growth factor-D (VEGF-D) correlates with up-regulated lymphangiogenesis and regional lymph node metastasis. Here, we have used the Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis to investigate the functional role of VEGF-D in the induction of lymphangiogenesis and tumor progression. Expression of VEGF-D in beta cells of single-transgenic Rip1VEGF-D mice resulted in the formation of peri-insular lymphatic lacunae, often containing leukocyte accumulations and blood hemorrhages. When these mice were crossed to Rip1Tag2 mice, VEGF-D-expressing tumors also exhibited peritumoral lymphangiogenesis with lymphocyte accumulations and hemorrhages, and they frequently developed lymph node and lung metastases. Notably, tumor outgrowth and blood microvessel density were significantly reduced in VEGF-D-expressing tumors. Our results demonstrate that VEGF-D induces lymphangiogenesis, promotes metastasis to lymph nodes and lungs, and yet represses hemangiogenesis and tumor outgrowth. Because a comparable transgenic expression of vascular endothelial growth factor-C (VEGF-C) in Rip1Tag2 has been shown previously to provoke lymphangiogenesis and lymph node metastasis in the absence of any distant metastasis, leukocyte infiltration, or angiogenesis-suppressing effects, these results reveal further functional differences between VEGF-D and VEGF-C.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia

This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of fibroblast growth factor 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total 16 mg: 4 x 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.