Biomolecular markers and involution of hemangiomas

Background/purpose

Vascular anomalies are a diverse set of lesions with distinct clinical behaviors, whose biomolecular characteristics are largely undefined. Common hemangiomas proliferate during the first year of life, then involute at a variable pace over several years. Other vascular tumors may involute much more quickly (rapidly involuting congenital hemangiomas [RICH]), not at all (lymphatic malformation), or display malignant behavior (angiosarcoma). Key cytokines driving angiogenesis include vascular endothelial growth factor (VEGF) family members/receptors (placental growth factor [PIGF], VEGF-A, and VEGF-C) and angiopoietins. The authors hypothesized that involuting hemangiomas would display biologic markers distinctly different from noninvoluting vascular lesions.

Methods

Six patient samples were analyzed: (1) RICH, (2) proliferating hemangioma, (3) involuting hemangioma, (4) tufted angioma, (5) hepatic angiosarcoma, and (6) lymphatic malformation. Detailed examination of endothelial/vascular mural cell status was performed by fluorescent double-label immunostaining using specific markers (PECAM-1, αSMA) in combination with markers of proliferation (anti-phospho-histone H3) or apoptosis (TUNEL). Expression of PIGF, VEGF-A, VEGF-C, and Ang-1 was localized by in situ hybridization.

Results

Involuting/proliferating common hemangiomas demonstrated vasculature with abundant vascular mural cells (αSMA+); in contrast, αSMA(+) cells were rare in RICH vessels. Endothelial apoptosis was increased dramatically, but proliferation was unchanged during involution. VEGF-A was expressed in all lesions except lymphatic malformation, which displayed VEGF-C and Ang-1 upregulation. Strikingly, PIGF expression was increased markedly in the lesions predicted to involute/actively involuting but was virtually absent from noninvoluting tumors.

Conclusions

Vessel architecture and endothelial/vascular mural cell status differed between lesions, differentiating even common versus rapidly involuting hemangioma and corresponded to clinical involution. VEGF-A expression characterized endothelial-derived lesions, whereas VEGF-C marked lymphatic-derived cells. PIGF expression occurred only in vascular anomalies predicted to involute or actively involuting, a pattern potentially linked to PIGF function as a conditional antagonist of VEGF-A. Thus, distinct patterns of morphology and angiogenic factor expression characterize vascular anomalies with different clinical behaviors.     

In vitro characteristics of endothelium from hemangiomas and vascular malformations

Hemangiomas are vascular tumors characterized by rapid growth, increased endothelial turnover, and increased numbers of mast cells. Vascular malformations grow commensurately with the patient or expand secondary to hemodynamic alteration and are characterized by a normal endothelial cell cycle and normal numbers of mast cells. Operative specimens of vascular birthmarks were categorized as hemangiomas or malformations based on clinical history, light microscopic examination, and mast cell quantitation. The specimens were cultured in tumor-conditioned medium and plasma clot culture. Capillary endothelium derived from hemangioma specimens formed capillary tubes in tissue culture--"angiogenesis in vitro." Capillary endothelium from malformations was difficult to culture and was not observed to form tubules. Hemangioma specimens demonstrated rapid outgrowth of tubular structures from plasma clot cultures, whereas malformation tissue did not produce such outgrowth. These results indicate that hemangioma endothelium grows preferentially in culture in comparison with endothelium from vascular malformations. It suggests a biologic difference, which correlates with our previously proposed classification.     

Reinnervation after nerve suture in rat groin flaps

Regeneration of sensory and adrenergic nerves in the skin was studied in rats. The aim was to investigate the effect of reanastomosing the cut nerve ends of the nerve trunk leading to the microvascular groin flap. Reinnervation was demonstrated immunohistochemically using calcitonin gene-related peptide (CGRP) as marker for sensory nerves, neuropeptide Y (NPY) and tyrosine hydroxylase (TH) as markers for adrenergic nerves and Protein Gene Product 9.5 (PGP 9.5) as general neuronal marker. It was demonstrated that reanastomosing of the nerve trunk was favourable for both the sensory and sympathetic reinnervation of microsurgical flaps. © 1998 Wiley-Liss, Inc. MICROSURGERY 18:1–5, 1998.     

血管瘤组织中TRAIL蛋白及其mRNA的表达和意义

目的检测血管瘤组织中TRAIL(肿瘤坏死因子相关凋亡诱导配体)蛋白及其mRNA的表达,并探讨其意义。方法采用免疫组织化学和原位杂交方法检测血管瘤增殖期、退化期、血管畸形和正常皮肤组织中TRAIL蛋白和mRNA的表达。结果TRAIL蛋白和mRNA在血管瘤增殖期和退化期中的阳性表达率分别为45.45%(15/33)和78.57%(22/28),66.67%(22/33)和89.29%(25/28),血管畸形和正常皮肤组织中均为阴性。秩和检验分别为Hc=54.03,Hc=71.82,四者之间差异均有统计学意义(P<0.01),两两之间比较,增殖期与退化期之间差异均有统计学意义(P<0.01)。结论TRAIL可能通过诱导内皮细胞凋亡,促进血管瘤的消退。     

Early nerve regeneration after Achilles tendon rupture — a prerequisite for healing? A study in the rat

Nerve regeneration during healing of Achilles tendon rupture in the rat was studied by immunohistochemistry including semi-quantitative assessment. Neuronal markers for regenerating and mature fibers, ie., growth associated protein 43 (GAP-43) and protein gene product 9.5 (PGP 9.5), respectively, were analyzed at different time points (1-16 weeks) post-rupture. In the paratenon, both the ruptured and intact contralateral tendon (control) consistently exhibited immunoreactivity to the two neuronal markers. However, in the proper tendinous tissue only the ruptured tendon showed immunoreactivity to GAP-43 and PGP 9.5. This expression was seen already at week 1 post-rupture to reach a peak at week 6 followed by a successive drop till week 16. Also the occurrence of sensory and autonomic fibers according to immunoreactivity for calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY), respectively, was analyzed. CGRP-positivity was abundantly seen from weeks 2-6 in both perivascular and sprouting free nerve endings in the proper tendon tissue undergoing healing. NPY appeared later, at weeks 6-8 post-rupture around blood vessels mainly located in the surrounding loose connective tissue. Apart from a role in vasoaction (CGRP, vasodilatory; NPY, vasoconstrictory). both neuropeptides have been implicated in fibroblast and endothelial cell proliferation required for angiogenesis. The present study shows that early healing of ruptured tendons is characterized by an orchestrated, temporal appearance of nerve fibers expressing peptides with different actions. The observed pattern of neuronal regeneration and neuropeptide expression may prove to be important for normal connective tissue healing.     

Morphological basis for back pain: the demonstration of nerve fibers and neuropeptides in the lumbar facet joint capsule but not in ligamentum flavum.

The innervation of lumbar facet capsule and ligamentum flavum was investigated using antisera to a general neuronal marker protein gene product (PGP) 9.5 and to peptide markers of sensory nerves (calcitonin gene-related peptide [CGRP] and substance P) and autonomic nerves (vasoactive intestinal polypeptide [VIP] and C-flanking peptide of neuropeptide Y [CPON]). In the facet capsule (n = 14), PGP 9.5 and CGRP-immunoreactive nerves occurred in 12 and five specimens, respectively, both around blood vessels and as free fibers in the stroma. Free fibers immunoreactive for substance P or VIP were noted in three and five specimens, whereas in nine specimens there were CPON-immunoreactive nerves located perivascularly. There was no immunoreactivity in the ligamentum flavum. This study provides further evidence that the facet capsule but not the ligamentum flavum has substantial innervation by sensory and autonomic nerve fibers and has a structural basis for pain perception.     

皮肤血管瘤和血管畸形细胞增殖与凋亡的对比研究

目的：探讨皮肤血管瘤和血管畸形的细胞凋亡与增殖的平衡关系及与临床生物学行为的可能关系。方法：采用流式细胞术检测皮肤血管瘤和血管畸形组织中凋亡细胞比率及细胞周期分布,分析细胞凋亡/增殖水平。结果：血管瘤增生期和消退期凋亡/增殖水平出现下调和上调（P〈0.05）,而血管畸形细胞凋亡/增殖水平与正常皮肤相比无显著性差异（P〉0.05）。结论：细胞凋亡/增殖水平在皮肤血管瘤和血管畸形之间存在差异且可能与二者的病理演变过程密切相关。     

Complications in circulation hamper the reinnervation of rat groin flaps

The reinnervation of rat groin island flaps and microneurovascular flaps was investigated. The nerve trunk leading to the flap was transsected in all rats and the nerve ends were either resutured or ligated. Sensory and adrenergic reinnervation of the flaps was studied semiquantitatively after 20 weeks using specific antisera for calcitonin gene-related peptide (CGRP) as a marker for sensory nerves, neuropeptide Y (NPY) for adrenergic nerves and Protein Gene Product 9.5 (PGP 9.5) as a general neuronal marker. The reinnervation of island groin flaps was compared to that in corresponding microneurovascular flaps. The nerve suture clearly increased both sensory and adrenergic reinnervation. In island flaps the reinnervation was good throughout the flap, whereas in microsurgical flaps the reinnervation pattern was more sparse and patchier. Evidently the cause for this was circulatory disorders and the reperfusion injury which takes place after the ischemic period in microsurgical flaps. Received: 3 March 1997 / Accepted: 14 January 1998     

The reinnervation pattern of wounds and scars may explain their sensory symptoms.

Anaesthesia, pruritis and pain are common in cutaneous scars. The reinnervation pattern of healing wounds and scars might help to explain these symptoms, as sensory neurotransmitters are known to be mediators of inflammation and healing. We quantified the regeneration patterns of blood vessels and nerves in excisional skin wounds as they matured into scars. Mice underwent 1cm(2) full thickness skin excisions. Wounds were harvested between five and 84 days. Sections underwent immunohistochemical staining for protein gene product 9.5 (PGP9.5) a pan-neuronal marker, and the sensory neuropeptides calcitonin gene related peptide (CGRP) and substance P (SP). The endothelial marker von Willebrand factor (VWF) was used to allow co-localisation and quantification of blood vessels. Nerve fibre density was quantified at multiple sites within wounds. There was no difference in the reinnervation/revascularisation pattern between peripheral and central sites. The density of PGP9.5, CGRP, SP and VWF peaked between 14 and 42 days, and levels of PGP9.5, CGRP and VWF all decreased to approximately those found in unwounded skin by 84 days (mature scar). SP levels, however, remained elevated at approximately twice the density found in unwounded skin. Increased densities of SP and CGRP in healing wounds could explain the unpleasant sensory symptoms of healing wounds.     

Silver impregnation and immunohistochemical study of nerves in lumbar facet joint plical tissue

Impingement of plical synovial tissue in a facet joint could cause pain. Plical tissue was removed during surgery for recurrent disc herniation or spinal stenosis. The presence of nerves was studied with silver impregnation, immunofluorescence, and avidin-biotin-peroxidase complex (ABC) immunostaining. Heterologous antisera to protein gene product (PGP) 9.5, substance P, calcitonin gene-related peptide (CGRP), and galanin were used to stain nerves. After silver impregnation, nerve-like structures were observed perivascularly. Such nerves located close to blood vessels were also immunoreactive for PGP 9.5, a more general cytoplasmic neural marker, whereas only few perivascular small varicosities were seen with antisera to substance P and galanin and none with antiserum to CGRP. In addition, PGP-9.5-, substance-P-, and galanin-immunoreactive nerves were occasionally seen very near to fat globules. Very few peptide-immunoreactive nerve varicosities were seen with immunofluorescence, and none of the PGP-9.5-immunoreactive nerves that were observed with ABC immunostaining were immunoreactive for neuropeptides as well. One mechanism for pain production could be mechanical compression of fatty tissue, but it is considered more likely that nerves in this particular tissue are mainly involved in local vasoregulation and that they are not sensory nociceptive nerves.     

尿碱性成纤维细胞生长因子用于鉴别血管瘤和血管畸形的作用

目的 探讨尿碱性成纤维细胞生长因子（bFGF）用于鉴别血管瘤和血管畸形、判断血管瘤是处于增生期或消退期以及动态监测血管瘤病程的作用。方法 对133例患儿（包括增生期血管瘤69例、消退期血管瘤41例及血管畸形23例）,应用酶联免疫吸附试验,检测尿bFGF浓度,并以无上述血管病变的唇腭裂11例患儿作为对照。结果 增生期血管瘤患儿尿bFGF浓度显著高于消退期血管瘤、血管畸形患儿和对照患儿。前3者与对照患儿比较,差异均有统计学意义（P〈0．01）：后3者之间两两比较,差别均无统计学意义（P〉0．05）。结论 尿bFGF浓度有助于鉴别血管瘤和血管畸形;并可判断血管瘤是处于增生或消退期及动态监测血管瘤病程,可为分析血管瘤的发生机制提供依据。     

Glut-1和VEGF在血管瘤和血管畸形中的表达及意义

目的：检测萄萄糖转运蛋白-1（Glut-1）和血管内皮细胞生长因子（VEGF）在血管瘤和血管畸形中的表达,探讨他们在血管瘤发生发展过程中的作用。方法：采用免疫组化SABC法检测30例增殖期血管瘤,30例消退期血管瘤,22例血管畸形（10例蔓状血管瘤和12例海绵状血管瘤）及5例正常皮肤组织中Glut-1和VEGF的表达。结果：增殖期血管瘤中Glut-1、VEGF的表达均显著高于消退期血管瘤、血管畸形和正常皮肤（P〈0．01）,消退期血管瘤中Glut-1的表达也明显高于血管畸形和正常皮肤（P〈0．01）,而消退期血管瘤中VEGF的表达与血管畸形,正常皮肤的差异不具有显著性（P〉0．05）。结论：Glut-1和VEGF在血管瘤的发生发展中可能起到很重要的作用,通过检测Glut-1在血管瘤和血管畸形中的表达情况能够比较准确的区分血管瘤和血管畸形,Glut-1或许可以作为区分血管瘤和血管畸形的一种较特异的参考指标。     

降钙素基因相关肽对人脐静脉血管内皮细胞增殖和迁移的影响及机制研究

目的组织工程骨的神经化能有效促进支架材料内血管生成,修复骨缺损。研究降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖与迁移的作用,进一步揭示组织工程骨的神经化促血管生成机制。方法体外分离获取HUVECs,并通过血管性血友病因子(von Willebrand factor,vWF)与CD31抗原鉴定,取第1代细胞用于实验。实验分为6组,分别以0(A组)、1×10—12(B组)、1×10—11(C组)、1×10—10(D组)、1×10—9(E组)、1×10—8mol/L(F组)浓度CGRP干预HUVECs。采用细胞免疫荧光观察HUVECs的CGRP1受体(CGRP1 receptor,CGRP1R)表达情况,AlarmarBlue法动态检测各组HUVECs增殖率,Transwell小室检测各组HUVECs的迁移能力,ELISA法检测HUVECs分泌VEGF的水平,Westernblot法检测其局部黏着斑激酶(focal adhesion kinase,FAK)的表达。结果分离的细胞通过形态学及vWF、CD31免疫荧光鉴定为HUVECs,并可见CGRP1R在细胞质和细胞膜表达。CGRP呈时间-浓度依赖性刺激HUVECs增殖;B～F组各时间点细胞增殖能力均高于A组(P<0.05),F组各时间点细胞增殖能力最高。B～F组迁移细胞数均显著高于A组(P<0.05),最大增幅达3倍以上。B～F组VEGF分泌量均显著高于A组(P<0.05);C、D组促进细胞分泌VEGF的能力最强。Western blot检测示,与A组相比,B～F组CGRP刺激HUVECs 3、7、10 d后,FAK表达明显增加(P<0.05)。结论 CGRP对HUVECs的增殖和迁移有直接促进作用,可能作用机制为CGRP能促进VEGF分泌和增加FAK的表达。     

Intratemporal facial nerve hemangiomas

Facial nerve hemangiomas are benign vascular tumors that arise within the temporal bone and have a histologic appearance similar to both cavernous hemangiomas and vascular malformations. In contrast to facial nerve schwannomas, these are extraneural tumors that cause symptoms by compression and tend to produce deficits when very small in size. We report our experience at the House Ear Clinic with 34 patients having these nonglomus intratemporal vascular tumors. Hemangiomas arising in the internal auditory canal tend to produce a progressive sensorineural hearing loss and are demonstrated with magnetic resonance imaging (MRI), whereas those at the geniculate ganglion are usually first seen with facial nerve symptoms and may require high-resolution computerized tomography (CT) for detection. Facial electromyography is helpful in establishing the diagnosis. Because of their extraneural nature, early diagnosis can permit removal of the tumor with preservation of facial nerves in some patients.     

Bcl-2和Bax在皮肤血管瘤组织中的表达及意义

目的 探讨Bcl-2和Bax在皮肤血管瘤发生、发展及退化过程中的作用及意义.方法 采用免疫组织化学方法(S-P法)检测人皮肤血管瘤增生期、退化期及正常组织中Bcl-2和Bax的表达水平.结果 Bcl-2在增生期血管瘤内皮细胞的表达明显高于退化期血管瘤内皮细胞和正常皮肤组织血管内皮细胞(P＜0.01);Bcl-2在退化期血管瘤内皮细胞的表达与正常皮肤组织血管内皮细胞相比,差异无统计学意义(P＞0.05).Bax在退化期血管瘤内皮细胞的表达明显高于增生期血管瘤内皮细胞和正常皮肤组织血管内皮细胞(P＜0.01);Bax在增生期血管瘤内皮细胞的表达高于正常皮肤组织血管内皮细胞(P＜0.05).结论 Bcl-2和Bax参与了血管瘤的发生、发展和退化.Bcl-2通过抑制内皮细胞凋亡而促进血管瘤的增生.Bax通过诱导内皮细胞凋亡而促进血管瘤由增生向退化的转变.     

Mechanisms and targets of angiogenesis and nerve growth in osteoarthritis

During osteoarthritis (OA), angiogenesis is increased in the synovium, osteophytes and menisci and leads to ossification in osteophytes and the deep layers of articular cartilage. Angiogenic and antiangiogenic factors might both be upregulated in the osteoarthritic joint; however, vascular growth predominates, and the articular cartilage loses its resistance to vascularization. In addition, blood vessel growth is increased at--and disrupts--the osteochondral junction. Angiogenesis in this location is dependent on the creation of channels from subchondral bone spaces into noncalcified articular cartilage. Inflammation drives synovial angiogenesis through macrophage activation. Blood vessel and nerve growth are linked by common pathways that involve the release of proangiogenic factors, such as vascular endothelial growth factor, β-nerve growth factor and neuropeptides. Proangiogenic factors might also stimulate nerve growth, and molecules produced by vascular cells could both stimulate and guide nerve growth. As sensory nerves grow along new blood vessels in osteoarthritic joints, they eventually penetrate noncalcified articular cartilage, osteophytes and the inner regions of menisci. Angiogenesis could, therefore, contribute to structural damage and pain in OA and provide potential targets for new treatments.     

雌激素受体、血管内皮生长因子和碱性成纤维细胞生长因子在小儿血管瘤中的表达及意义

目的探讨雌激素在小儿血管瘤增殖退化过程中的作用。方法用免疫组织化学法检测42例血管瘤和17例血管畸形标本雌激素受体（estrogen receptor，ER）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、碱性成纤维细胞生长因子（basic fibroblast growth fictor，bF—GF）的表达，分析其相关性。结果血管瘤组的ER、VEGF、bFGF平均标记指数均显著高于血管畸形组及正常皮肤组（P《0．01），血管瘤组中增生期亚组三指标标记指数均显著高于退化期亚组（P〈0．01）。血管瘤中ER与VEGF、bFGF具强正相关性，其相关系数分别为0．91、0．83，而血管畸形组中该相关性较弱，相关系数分别为0．20、0．58（P〈0．05）。结论雌激素及其受体与小儿血管瘤血管生成密切相关。     

Noninvasive monitoring of diabetes-induced cutaneous nerve fiber loss and hypoalgesia in thy1-YFP transgenic mice

Progressive loss of pain perception and cutaneous nerve fibers are frequently observed in diabetic patients. We evaluated the feasibility of using thy1-YFP mice that express the yellowish-green fluorescent protein (YFP) in all of their sensory/motor neurons for noninvasive monitoring of cutaneous nerve fiber loss during diabetes. Fluorescent fibers in skin sections from the leg of thy1-YFP mice stained positive for the neuron-specific protein gene product 9.5 (PGP9.5), indicating that the cutaneous fluorescent fibers are indeed nerve fibers. In diabetic thy1-YFP mice, significant small cutaneous nerve fiber loss in the leg was observed at 3 months following the onset of diabetes, but loss of heat-induced pain perception occurred as early as 1 month following the onset of diabetes, indicating that functional impairment of sensory nerves precedes cutaneous nerve fiber loss. Immunostaining of skin sections of mice killed at 6 months following the onset of diabetes showed that parallel to the loss of small fluorescent nerve fibers, there was a significant decrease in fibers stained positive for calcitonin gene-related peptide, substance P, and purinoreceptor subtype in diabetic thy1-YFP mice. These mice will be useful for noninvasive monitoring of cutaneous nerve fiber degeneration and loss of heat-induced pain perception during diabetes and for the assessment of efficacy of therapeutic treatment of diabetic neuropathy.     

Neuronal regeneration in denervated muscle following sensory and muscular neurotization

Background and purpose?Neurotization of denervated muscles has been shown to improve muscle bulk, but the neuronal regeneration response has not been compared previously in different surgical techniques of neurotization. Thus, using a rat model of experimental skeletal muscle denervation, we studied neuronal regeneration following sensory neurotization by two methods: sensory nerve to motor branch of muscle and direct sensory nerve implantation to muscle.

Material and methods?The lateral head of the gas-trocnemius muscle was denervated in 36 rats, of which the first 12 served as denervated controls. In the second group of 12, the sural nerve was anastomozed to the motor branch of the gastrocnemius muscle (sensory-to-motor nerve neurotization) and in the remaining 12 rats the sural nerve was split into 4 fascicles and embedded into 4 quadrants of the muscle (direct sensory nerve-to-muscle neurotization). Immunohistochemistry was used to examine nerve fibers in muscle containing the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and general neuronal marker protein gene product 9.5 (PGP 9.5).

Results?Semiquantitative analysis showed that, compared to the control side, the number of nerve fibers on the experimental side was highest (p < 0.01) for group III (direct sensory nerve-to-muscle neurotization) for all 3 markers. The difference was 71%, 298%, and 254% for PGP 9.5, CGRP, and SP, respectively.

Interpretation?This method may be a good option for inducing neuronal regeneration in denervated muscles, and has therapeutic implications for prevention of atrophy of denervated muscles and as an adjunct for reconstruction of soft tissue defects.