Combination of platelet-derived growth factor-BB and insulin-like growth factor-I is more effective than platelet-derived growth factor-BB alone in stimulating complete healing of full-thickness wounds in "older" diabetic mice

Platelet-derived growth factor and insulin-like growth factor-I have been shown to interact synergistically to enhance repair of skin wounds in normal healing swine. Platelet-derived growth factor alone has shown promise in treating human chronic ulcers. The objective of this study was to compare the wound healing effects of platelet-derived growth factor-BB alone with those of a combination of platelet-derived growth factor-BB and insulin-like growth factor-I in an improved model with the use of "older" animals with diabetes. Older diabetic (db/db) mice (>15 weeks of age) have less elevated insulin levels compared with young db/db mice. The serum insulin levels in the older animals is 1.0 to 2.5 times that of the nondiabetic animals, a similar increase to that which occurs in human patients with type II diabetes. Healing was evaluated in two studies involving a total of 104 animals. Treatment groups included the following: 4.0 microg/cm(2) of platelet-derived growth factor-BB, 40.0 microg/cm(2) of platelet-derived growth factor-BB, 4.0 microg/cm(2) of both platelet-derived growth factor-BB and insulin-like growth factor-I or vehicle. All growth factors were applied topically in a methylcellulose vehicle to full-thickness wounds every other day for 24 days. Efficacy end points were median and mean time to complete healing and rate of wound closure. The median time to complete healing for animals receiving the platelet-derived growth factor-BB/insulin-like growth factor-I combination was 38% and 33% faster (p < 0.001) than animals receiving 4.0 microg/cm(2) and 40.0 microg/cm(2) of platelet-derived growth factor-BB, respectively. The mean time to complete healing for platelet-derived growth factor/insulin-like growth factor-I treated animals was 31% and 29% faster (p < 0.001) than 4.0 microg/cm(2) and 40.0 microg/cm(2) platelet-derived growth factor-BB treated animals, respectively. Wounds treated with 4.0 microg/cm(2) platelet-derived growth factor-BB/insulin-like growth factor-I healed, on average, in 22 days compared with 31 days for 40.0 microg/cm(2) platelet-derived growth factor-BB alone and 38 days for vehicle. Also, platelet-derived growth factor-BB/insulin-like growth factor-I significantly improved the rate of wound closure throughout the duration of the studies compared with either dose of platelet-derived growth factor-BB alone (p < 0.005) or vehicle (p < 0.001). In conclusion, the data show that the combination of platelet-derived growth factor-BB and insulin-like growth factor-I is more effective than platelet-derived growth factor-BB alone at the doses tested or vehicle treatment in stimulating cutaneous wound healing in older, diabetic mice.     

血小板衍生生长因子－BB对破骨细胞功能影响的实验研究

目的探讨血小板衍生生长因子 -BB(platelet-derivedgrowthfactor-BB,PDGF -BB)对破骨细胞生物学功能的影响。方法 (1)利用酶消化法分离培养成人破骨细胞 ;(2)应用透射电子显微镜方法观察破骨细胞对PDGF -β 受体的表达 ;(3)在经过纯化的破骨细胞上清液中施加重组人基因PDGF -BB ,利用酶动力学方法测定培养上清液中的酸性磷酸酶 (ACP)和抗酒石酸酸性磷酸酶(TRAP)的活性 ;(4)通过Leica图像分析仪观察在PDGF -BB作用下 ,破骨细胞所形成骨吸收陷窝的数目与面积。结果 (1)破骨细胞膜上有胶体金颗粒沉着 ;(2)破骨细胞培养上清液中的ACP和TRAP活性随着PDGF -BB浓度的升高而增加 ,分别从(1.85±0.13)u/L和(1.73±0.15)u/L(对照组 )增加至(2.86±0.15)u/L和(2.75±0.24)u/L(40μg/L组 ,P<0.01) ;(3)在PDGF -BB的作用下 ,破骨细胞所形成的骨吸收陷窝的面积从(435.08±237.50)μm2(对照组 )增高至(630.26±240.64)μm2 (40μg/L组 ,P<0.01) ,每个骨片上骨吸收陷窝的数目亦从14.00±1.41增加为26.00±2.00(P<0.05或P<0.01)。结论PDGF -BB通过与PDGF -β受体结合 ,可直接促进破骨细胞的骨吸收功能     

Low-intensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-I expression in rat calvarial cells

BACKGROUND AND OBJECTIVE: We previously reported that low-intensity laser irradiation stimulated bone nodule formation through enhanced cellular proliferation and differentiation. However, the mechanisms of irradiation are unclear. Thus, we attempted to determine the responsibility of insulin-like growth factor (IGF)-I for the action observed. STUDY DESIGN/MATERIALS AND METHODS: Osteoblast-like cells were isolated from fetal rat calvariae and cultured with rat recombinant (r) IGF-I, IGF-I-antibody (Ab), and/or the cells were irradiated once (3.75 J/cm(2)) with a low-intensity Ga-Al-As laser (830 nm). The number and area of bone nodules formed in the culture were analyzed, and IGF-I expression was also examined. RESULTS: Treatment with rIGF-I significantly stimulated the number and area of bone nodules. This stimulatory effect was quite similar to those by laser irradiation, and this stimulation was abrogated dose-dependently by treatment with IGF-I-Ab. Moreover, laser irradiation significantly increased IGF-I protein and gene expression. CONCLUSION: The stimulatory effect of bone nodule formation by low-intensity laser irradiation will be at least partly mediated by IGF-I expression.     

Growth hormone but not insulin-like growth factor-I improves wound strength in pigs

Systemic growth hormone and locally administered insulin-like growth factor-I have been shown in a number of studies to improve the breaking strength of incisional wounds, especially in compromised animals. The objective of the present study was to compare these two agents when administered subcutaneously distant from an incisional wound site in pigs, as well as to examine effects of a combination growth hormone/insulin-like growth factor treatment. Growth hormone was shown to increase wound breaking strength in two experiments, whereas insulin-like growth factor-I or a more potent analog had no effect. Moreover, breaking strength was only minimally improved above the vehicle groups by the combination of growth hormone and insulin-like growth factor-I. These effects could not be explained by changes in plasma insulin-like growth factor-I concentrations which were highest in the combination groups, nor by plasma insulin-like growth factor binding protein-3 which was raised equally whenever growth hormone was administered. We conclude that systemic growth hormone but not insulin-like growth factor-I improves wound strength in normal pigs, whereas insulin-like growth factor-I reduces the magnitude of the growth hormone effect by an unknown mechanism.     

Triiodothyronine stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

We examined the effects of triiodothyronine (T₃) on⁴⁵Ca-accumulation into extracellular matrix in osteoblast-like MC3T3-El cells cultured for 7-week period, which was considered to be long enough to develop calcification. T₃ increased⁴⁵Ca-accumulation into sodium dodecyl sulfate-insoluble, EDTA-extractable materials cultures of MC3T3-El cells in a dose-dependent manner in the range between 10 pM and 10 nM. Contrary to the stimulatory effect on⁴⁵Ca-accumulation, T₃ inhibited DNA synthesis in MC3T3-El cells dose dependently between 10 pM and 10 nM. T₃ stimulated the secretion of insulin-like growth factor-I (IGF-I) dose dependently between 0.1 nM and 10 nM in MC3T3-El cells cultured for 7 weeks. Antibodies to IGF-I suppressed the⁴⁵Ca-accumulation induced by T₃ almost to the control level. These results strongly suggest that T₃ stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Cyclic AMP stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

Summary The effects of cAMP on insulin-like growth factor-I (IGF-I) secretion and⁴⁵Ca-accumulation into extracellular matrix were investigated in cloned osteoblast-like MC3T3-E1 cells cultured for 7 weeks, which was considered to be long enough to develop calcification. Dibutyryl cAMP stimulated IGF-I secretion dose dependently in the range between 0.1 mM and 3 mM. Dibutyryl cAMP also stimulated accumulation of⁴⁵Ca into sodium dodecyl sulfate-insoluble, EDTA-extractable materials in cultures of these cells dose dependently between 0.1 mM and 3 mM. The patterns of both stimulations were similar. The dibutyryl cAMP-stimulated IGF-I secretion preceded the⁴⁵Ca-accumulation. Antibodies to IGF-I suppressed the dibutyryl cAMP-stimulated⁴⁵Ca-accumulation almost to the control level. Taking account of our previous report that exogenous IGF-I stimulates⁴⁵Ca-accumulation in cultures of these cells, the present results suggest that cAMP stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Recombinant human insulin-like growth factor-I stimulates in vitro matrix synthesis and cell proliferation in rabbit flexor tendon

Flexor tendons have an intrinsic ability for repair, with a capacity to metabolize matrix components and to proliferate. To identify factors with the potential of affecting those abilities, the effects of recombinant human insulin-like growth factor (rhIGF-I), insulin and fetal calf serum (FCS) on the synthesis of proteoglycan, collagen, and non-collagen protein and cell proliferation were investigated in short-term explant cultures of the deep flexor tendon of the rabbit. Matrix synthesis and cell proliferation were stimulated dose dependently by rhIGF-I at doses between 10 and 250 and at 10-100 ng/ml, respectively, by insulin at 250-5,000 ng/ml, and by FCS at 2-15%. Estimated maximal stimulation (Emax) of up to three times the control value was observed with rhIGF-I at 250 ng/ml. Maximal stimulation was observed at 5,000 ng/ml with insulin, and FCS at 15%. rhIGF-I was more potent than insulin in stimulating protein synthesis and cell proliferation. The Emax of stimulation of proteoglycan and collagen synthesis by rhIGF-I were two times that of FCS, and the Emax of cell proliferation by FCS was twice that of rhIGF-I. Growth factors thus have the ability to stimulate matrix synthesis and cell proliferation in rabbit flexor tendon. This provides a rationale for further studies on the role of growth factors in flexor tendon healing in humans.     

The contribution of platelet-derived growth factor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation of osteoblast-like cells in vitro. PRP was prepared using a centrifuge; the number of platelets (n = 32) and the levels of platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-I (IGF-I) were measured (n = 16). For the proliferation assay, SaOS-2 was cultured in the presence of platelet-poor plasma (PPP), whole blood, or PRP. The cell number was counted after 36 and 72 hours. To investigate the effect of each growth factor, the cells were cultured with PRP in the absence or presence of neutralizing antibodies, and counted as described. The mean platelet count of PRP was 1546.36 +/- 382.25 x 10(3)/microL, and the mean levels of PDGF-AB, TGF-beta1 and IGF-I were 0.271 +/- 0.043, 0.190 +/- 0.039, and 0.110 +/- 0.039 ng/1500 x 10(3) platelets, respectively. Cell proliferation was enhanced in all PRP groups in a dose-dependent manner, and all neutralizing antibodies significantly suppressed proliferation compared with the PRP group, lacking antibody, at 36 hours. However, at 72 hours, the neutralizing antibodies of PDGF and TGF-beta1, but not IGF-I, significantly suppressed proliferation. These results show the beneficial abilities of PRP in the proliferation of osteoblast-like cells from the standpoint of growth factors, including the contribution of each factor.     

The role of growth hormone and insulin-like growth factor-I in experimental renal growth and scarring

Recent evidence suggests a causal link between early renal/glomerular hypertrophy and late kidney scarring and glomerular sclerosis. Insulin-like growth factor-I (IGF-I) is a growth-promoting peptide likely to play a role in the development of kidney growth. We observed an increased renal IGF-I content in two experimental models of accelerated kidney growth in the rat. By contrast, diabetic renal hypertrophy is abolished in the absence of growth hormone (GH). Dietary protein manipulations affect the expression of compensatory renal growth (CRG), as well as renal IGF-I content. The renotrophic effect of a high-protein diet on CRG seems GH-dependent and IGF-I-mediated. GH also appears to have a permissive role on the development of progressive renal scarring following extensive renal ablation in rats, as dwarf rats seem somewhat resistant to the development of accelerated scarring and renal failure.     

Reversal of impaired wound healing in irradiated rats by platelet-derived growth factor-BB

This study examined the potential influence of platelet-derived growth factor-BB homodimers (PDGF-BB) on surgical incisions in irradiated animals with depressed wound healing. Rats were irradiated with either 800 rads total body or 2,500 rads surface irradiation. Parallel dorsal skin incisions were made 2 days later, and PDGF-BB was applied topically a single time to one of two incisions. In total body-irradiated rats, bone marrow-derived elements were severely depressed, wound macrophages were virtually eliminated, and PDGF-BB treatment was ineffective. However, in surface-irradiated rats, PDGF-BB treatment recruited macrophages into wounds and partially reversed impaired healing on day 7 (p less than 0.005) and day 12 (p less than 0.001). PDGF-BB-treated wounds were 50 percent stronger than the paired control wounds. The results suggest PDGF requires bone marrow-derived cells, likely wound macrophages, for activity and that it may be useful as a topical agent in postirradiation surgical incisions.     

血小板源性生长因子BB对体外培养肌腱细胞增殖的影响

Objective To study the effect of platelet-derived growth factor-BB (PDGF-BB) in dif-ferent concentrations on proliferation of tendon cells cultured in vitro. Methods Rat tendon cells were cultured and identified in vitro. The rat tendon cells were cultured in PDGF-BB nutrient solution in different concentrations. They were then divided into 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB groups (cultured with 0.1 mL 0.5% PBS with addition of 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB respectively). Tendon cells in control group were cultured with 0.1 mL 0.5% FBS. Proliferation of tendon cells was detected by MTT test. The absorbance values of tendon cells in control group and 20 ng/mL PDGF-BB group before culture and after cultured for 12, 24, 36, 48, 60, 72 hs were determined. Results The isolated cells were identified to be rat tendon cells as they were Type Ⅰ collagen staining posi-tive and Type Ⅲ collagen staining negative. Compared with that of control group, the absorbance values of other groups were all increased, except for that of 250 ng/mL PDGF-BB group (P<0.05 or P<0.01). Besides, the absorbance value rose gradually with the increase of the concentration of PDGF-BB on, and then diminished gradually with the increase of the concentration of PDGF-BB from 20 ng/mL on. Tendon cells in 20 ng/ml PDGF-BB group began to increase in number when cultured for 12 hs, and it reached the highest level (0.53±0.04) at 48 h, which were obviously higher than those of control group at 24-72 h (P<0.01). The absorbance value of tendon cells in 20 ng/mL PDGF-BB group was significantly higher than that of control group at 24, 36, 48, 60, 72 h after culture (P<0.01 ). Conclusions PDGF-BB can promote the proliferation of tendon cells in a definite range of concentration and time.     

血小板源性生长因子BB对大鼠肌腱愈合和肌腱粘连的影响

目的 了解血小板源性生长因子BB(PDGF-BB)基因转染大鼠肌腱细胞对肌腱愈合及肌腱粘连的影响. 方法 将90只SD大鼠制成跟腱损伤模型,按随机数字表法分为3组,每组30只:实验组,肌腱断端注射20μL转染PDGF-BB基因的大鼠肌腱细胞(1×108个/mL);对照组,肌腱断端注射20μL未行转染的大鼠肌腱细胞(1×108个/mL);空白对照组,不做任何处理.6-0丝线行改良Kessler法缝合跟腱,管型石膏固定1周.通过基因测序及RT-PCR鉴定转染PDGF-BB基因的大鼠肌腱细胞.分别于术后3 d和1、2、4、8周取各组大鼠肌腱组织样本,行大体、组织学观察以及生物力学检测,对比各组肌腱粘连度、组织中Fb数量与胶原纤维含量、肌腱最大抗拉力及最大滑动距离、组织中PDGF-BB的浓度.对数据行t检验. 结果(1)转染的肌腱细胞经RT-PCR以及基因测序证实在体外稳定表达PDGF-BB mRNA.(2)各组大鼠术后3 d肌腱均出现较明显肿胀及炎性细胞浸润,实验组改变较其他组明显轻微;随后各组情况均逐渐好转.术后4、8周肌腱粘连度分级组间比较未见明显差异.(3)实验组Fb数在术后2、4、8周显著低于对照组和空白对照组(t值分别为2.94、4.26、5.76和4.00、3.83、6.12,P＜0.05或P＜0.01).(4)实验组术后4周胶原纤维含量为(43±6)%,较对照组[(55±8)%]与空白对照组[(61±8)%]显著下降(t值分别为2.94和4.41,P＜0.05或P＜0.01).(5)术后4、8周实验组肌腱最大滑动距离为(3.25±0.33)、(3.65±0.21)mm,显著高于对照组的(2.29±0.40)、(2.21±0.37)mm和空白对照组的(2.01±0.23)、(1.89±0.24)mm(t值分别为4.53、8.29和7.55、13.52,P值均小于0.01),但其肌腱最大抗拉力与另2组比较差异无统计学意义(t值分别为0.41、0.41和0.77、0.72,P值均大于0.05).(6)术后3 d和2、4周,实验组肌腱组织中PDGF-BB浓度为(12.95±1.36)、(8.32±0.94)、(9.10±1.06)ng/mL,均显著高于对照组的(1.13±0.21)、(2.07±0.48)、(3.85±0.39)ng/mL(t值分别为21.04、14.50、11.39,P值均小于0.01). 结论 转染PDGF-BB基因肌腱细胞有促进肌腱内源性愈合、减轻肌腱粘连的作用.     

Potentiation by platelet-derived growth factor-BB of FGF-2-stimulated VEGF release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of platelet-derived growth factor-BB (PDGF-BB) on FGF-2-induced VEGF release in MC3T3-E1 cells. PDGF-BB significantly enhanced the FGF-2-stimulated VEGF release. The amplifying effect of PDGF-BB was dose dependent in the range between 0.1 and 30 ng/ml. AG1295, a selective inhibitor of PDGF receptor kinase, which reduced the autophosphorylation of PDGF receptor-(R), suppressed the enhancement by PDGF-BB without affecting the FGF-2 effect. PDGF-BB failed to strengthen the FGF-2-induced phosphorylation of p44/p42 MAP kinase or SAPK/JNK. The amplification by PDGF-BB of FGF-2-stimulated VEGF release was reduced by PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK. These results strongly suggest that PDGF-BB potentiates FGF-2-stimulated VEGF release at a point downstream from p44/p42 MAP kinase and SAPK/JNK in osteoblasts.     

Compensatory renal growth: role of growth hormone and insulin-like growth factor-I

Recent experimental evidence suggests that insulin-like growth factor-I (IGF-I) may be involved in compensatory renal growth (CRG). This study was designed to determine the relative contribution of IGF-I and growth hormone (GH) to the CRG that takes place in rats following uninephrectomy (UNx). We also studied the respective role of GH and IGF-I in the stimulation of CRG induced by a high protein diet (HPD). CRG was studied 7 days after UNx in Wistar rats and in a new mutant strain of dwarf rats, selectively deficient in GH. Prior to UNx, rats of both strains were pre-fed (14 days) either a medium-protein diet (MPD, casein 18%) or a HPD (54%). On MPD, CRG was comparable in Wistar (17.6 +/- 3.1%, M +/- SD) and dwarf (14.4 +/- 4.8%) rats. The HPD enhanced CRG in the Wistars (27 +/- 3.9%, P less than 0.005) but not in the dwarfs (14.9 +/- 2%). CRG in both experimental groups involved renal hypertrophy and hyperplasia. Control (baseline) serum, liver and kidney IGF-I were significantly less in dwarf rats. However, following UNx, on a MPD, kidney IGF-I increased significantly in both Wistar and dwarf rats: Wistar, pre-UNx, 310 +/- 46 ng/g tissue; post-UNx, 405 +/- 54 ng/g, P less than 0.005; dwarfs, pre-UNx, 205 +/- 35 ng/g; post-UNx 426 +/- 90 ng/g, P less than 0.001. On a HPD a further significant increase in renal IGF-I was only observed in Wistar rats (505 +/- 46 ng/g). No change in serum or liver IGF-I was observed after UNx in either strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factor-I and insulin-like growth factor-binding proteins in the nephrotic syndrome

Similar to findings in the nephrotic syndrome in humans, rats with the doxorubicin-induced nephrotic syndrome (which resembles minimal change disease) have reduced serum levels of insulin-like growth factor-I (IGF-I). This is mainly caused by glomerular ultrafiltration of IGF-I-containing binding protein complexes, primarily of a molecular weight of approximately 50 kilodaltons, and urinary losses of the peptide. Despite urinary excretion of IGF-binding protein (IGFBP)-2, serum levels are increased more than twofold in the nephrotic syndrome compared with controls, because of increased synthesis of this binding protein by the liver. In contrast, the liver synthesis of IGFBP-3, the predominant binding protein in normal serum, is unchanged in the nephrotic syndrome. However, binding and serum levels of IGFBP-3 are reduced in nephrotic rat serum, apparently due to proteolytic degradation of IGFBP-3. The glomerular ultrafiltration of IGF-I, which leads to biologically significant IGF-I concentrations of about 1.35 nM in proximal tubule fluid, may have metabolic consequences, such as increased tubular phosphate absorption. Hypothetically, tubule fluid IGF-I may also contribute to progressive tubulointerstitial fibrosis which is sometimes present in protractive nephrotic glomerulopathies. The profound changes in the IGF-I/IGFBP system in the nephrotic syndrome may also contribute to systemic metabolic abnormalities and growth failure.     

The growth hormone/insulin-like growth factor-I system: implications for organ growth and development

Growth hormone (GH) and insulin-like growth factors (IGFs) are essential for normal growth and development during embryonic stages as well as postnatally. While GH has little effect on these processes prenatally, the IGFs are important during these stages. On the other hand the GH-IGF-I axis is important for pubertal growth. To determine whether postnatal growth and development are dependent on circulating or locally produced IGF-I, we deleted the IGF-I gene in the liver using the cre/LoxP system used for tissue-specific gene deletion. These animals demonstrated approximately 75%–80% reduction in circulating IGF-I and an approximate fourfold increase in circulating GH. Despite the marked reductions in circulating IGF-I, growth and development was apparently normal. Thus the original somatomedin hypothesis needs to be re-evaluated in the light of these new findings. Received: 5 September 1999 / Revised: 11 December 1999 / Accepted: 18 December 1999