水通道 AQP1 敲除小鼠肿瘤血管生成障碍及肿瘤生长减缓

血管生成是肿瘤生长、浸润和转移的必要步骤. 肿瘤血管生成涉及瘤旁组织血管内皮细胞增殖、向肿瘤细胞团内迁移以及管腔形成,目前机理尚不完全清楚. 水通道 AQP1 在多种肿瘤血管内皮高表达,提示其可能参与肿瘤血管的生成过程. 应用 AQP1 敲除小鼠荷瘤实验证实了 AQP1 在黑色素瘤生长和血管新生中的作用. 结果表明,皮下接种的黑色素瘤在 AQP1 敲除小鼠的生长较之在野生型小鼠延迟近 30％ (P<0.01). 免疫组化与肿 瘤病理形态学分析显示, AQP1 在野生型小鼠黑色素瘤血管内皮细胞上高表达,而在 AQP1 敲除小鼠黑色素瘤血管内皮细胞呈阴性表达. 在病理结构上,黑色素瘤细胞围绕血管分支呈岛状分布. 野生型小鼠黑色素瘤内血管管腔较细小,而 AQP1(－/－)小鼠黑色素瘤内血管床显著膨大. AQP1(－/－)小鼠肿瘤内平均微血管密度 (47/mm²) 较之 AQP1(＋/＋) 肿瘤 (142/mm²) 减少 67％ (P<0.01). 围绕 AQP1(－/－) 肿瘤血管的肿瘤细胞岛周边坏死区域明显大于 AQP1(＋/＋)肿瘤. 上述结果提出确切证据表明, AQP1 缺失使肿瘤血管生成发生障碍,从而影响了肿瘤血液供应和肿瘤生长. AQP1参与肿瘤血管生成的机理值得深入研究.     

Latent antithrombin does not affect physiological angiogenesis: an in vivo study on vascularization of grafted ovarian follicles

Latent antithrombin (L-AT), a heat-denatured form of native antithrombin (AT), is a potent inhibitor of pathological tumor angiogenesis. In the present study, we have investigated whether L-AT has comparable antiangiogenic effects on physiological angiogenesis of ovarian tissue. For this purpose, preovulatory follicles of Syrian golden hamsters were mechanically isolated and transplanted into dorsal skinfold chambers chronically implanted in L-AT- or AT-treated hamsters. Non-treated animals served as controls. Over 14 days after transplantation neovascularization of the follicular grafts was assessed in vivo by quantitative analysis of the newly developed microvascular network, its microvessel density, the diameter of the microvessels, their red blood cell velocity and volumetric blood flow as well as leukocyte-endothelial cell interaction using fluorescence microscopic techniques. In each group, all of the grafted follicles were able to induce angiogenesis. At day 3 after transplantation, sinusoidal sacculations and capillary sprouts could be observed, finally developing complete glomerulum-like microvascular networks within 5 to 7 days. Overall revascularization of grafted follicles did not differ between the groups studied. Interestingly, follicular grafts in L-AT- and AT-treated hamsters presented with higher values of microvessel diameters and volumetric blood flow, when compared to non-treated controls, which may be best interpreted as a reactive response to an increased release of vasoactive mediators. In conclusion, the present study demonstrates, that L-AT has no adverse effects on physiological angiogenesis of freely transplanted ovarian follicles. Thus, L-AT may be an effective drug in tumor therapy, which blocks tumor growth by selective suppression of tumor vascularization without affecting new vessel formation in the female reproductive system.     

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.     

Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis

VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.     

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.     

Expression of hepatocyte growth factor activator inhibitor type 1 in endothelial cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is an integral membrane Kunitz-type serine proteinase inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). HGFA is a serum proteinase that is critically involved in the activation of hepatocyte growth factor/scatter factor (HGF/SF) in injured tissue. Previous studies have shown that HAI-1 is expressed on the basolateral surface of various epithelial cells. In this study, we analyzed the expression of HAI-1 in human endothelial cells. Immunohistochemically, HAI-1 protein was observed in the endothelial cells of capillaries, venules and lymph vessels. On the other hand, arterial endothelial cells were poorly stained for HAI-1. Mesothelial cells on the serous surface were also positively immunostained. The endothelial expression of HAI-1 was also examined in cultured human endothelial cells of various origins, such as umbilical vein, microvessels and aorta. Notably, in accordance with the results of immunohistochemistry, HAI-1 mRNA and protein levels were high in the endothelial cells derived from umbilical vein and were hardly detectable in those derived from aorta. A low but distinct level of HAI-1 expression was also observed in endothelial cells from microvessels. As these HAI-1-positive endothelial cells also expressed MET tyrosine kinase, the specific receptor of HGF/SF, it is conceivable that HAI-1 might have an important regulatory role in the HGF/SF-MET signaling axis of endothelial cells, which could be involved in the process of angiogenesis.     

The role of fibroblast growth factors in vascular development

Fibroblast growth factors (FGFs) are considered angiogenic factors, yet the exact relationship between FGF and vascular development in normal and pathological tissue has long remained elusive. However, recent results from gene inactivation and transgenic studies in mice and in culture systems have demonstrated the role of FGFs in vessel assembly and sprouting. FGFs also promote blood-vessel branching and induce lymphangiogenesis. Novel players in FGF-mediated angiogenesis have been identified, such as p38 mitogen-activated protein kinase. Tumour angiogenesis is regulated by FGFs directly or indirectly via secondary angiogenesis factors, such as vascular endothelial growth factor. The newly established angiogenic role of FGFs makes FGF or molecules targeting FGF and its receptor promising candidates for the development of novel therapeutics.     

Role of vascular endothelial growth factor in ovarian physiology - an overview

In the female reproductive system, as in a few adult tissues, angiogenesis occurs as a normal process and is essential for normal tissue growth and development. In the ovary, new blood vessel formation facilitates oxygen, nutrients, and hormone substrate delivery, and also secures transfer of different hormones to targeted cells. Ovarian follicle and the corpus luteum (CL) have been shown to produce several angiogenic factors, however, vascular endothelial growth factor (VEGF) is thought to play a paramount role in the regulation of normal and abnormal angiogenesis in the ovary. Expression of VEGF in ovarian follicles depends on follicular size. Inhibition of VEGF expression results in decreased follicle angiogenesis and the lack of the development of mature antral follicles. The permeabilizing activity of VEGF is thought to be involved in follicle antrum formation and in the ovulatory process. In the CL, VEGF expression corresponds to different patterns of angiogenesis during its lifespan. In most the species, higher VEGF expression in the early luteal phase is essential for the development of a high-density capillary network in the CL. However, high VEGF expression may be still maintained in the mid-luteal phase to increase vascular permeability that results in enhancement of luteal function. During gestation, VEGF is thought to be important for the persistence of the CL function for a longer than in the nonfertile cycle period of time. Further elucidation of specific roles of VEGF in ovarian physiology may help to understand the phenomenon of luteal insufficiency and reveal novel strategies of ovarian angiogenesis manipulation to alleviate infertility or to control fertility.     

Innervation and neurokinin receptors during angiogenesis in the rat sponge granuloma

Summary Angiogenesis is an essential component of wound healing and inflammation. In the rat subcutaneous sponge implantation model, angiogenesis can be enhanced by administration of the sensory neuropeptide, substance P. We have used quantitativein vitro receptor autoradiography and immunohistochemistry to investigate the development of endogenous neurovascular regulatory systems in the newly-formed granulation tissue of this model. The fraction of endothelial cells immunoreactive for proliferating cell nuclear antigen, endothelial fractional area, and¹³³Xe clearance were used as measures of endothelial proliferation, neovascularization, and blood flow, respectively. Endothelial proliferation occurred predominantly in tissues surrounding the sponge, and peaked before neovascularization of sponge stroma and the establishment of sponge blood flow. Substance P-containing sensory nerves and specific, high affinity substance P binding sites with characteristics of neurokinin receptors of the NK₁ subclass, were localized to microvessels surrounding the sponge at all time points. Lower density substance P binding sites were localized to newly formed microvessels within the sponge stroma, progressively increasing in density from day 4 to day 14. Nerve fibres were observed in the stroma of only 2 of 6 sponges at day 14, and none at earlier time points. These data support the hypothesis that substance P-enhanced angiogenesis in this model results from a direct action on microvascular NK₁ receptors. Neovascularization is a sequential process, with early endothelial proliferation followed by new vessel formation and increased blood flow, with maturation of endogenous neurovascular regulatory systems occurring late in this process in inflamed tissues.     

Follicular fluid high density lipoprotein-associated sphingosine 1-phosphate is a novel mediator of ovarian angiogenesis

Angiogenesis plays an important role in the development of the ovarian follicle and its subsequent transition into the corpus luteum. Accordingly, follicular fluid is a rich source of mitogenic and angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor secreted by granulosa cells. In the present study, we show that follicular fluid deprived of basic fibroblast growth factor or vascular endothelial growth factor by means of thermal denaturation or antibody neutralization retains its capacity to stimulate endothelial proliferation and angiogenesis. Mass spectrometric analysis of chromatographic fractions stimulating endothelial growth obtained from follicular fluid revealed that the heat-stable mitogenic activity is identical with the subfraction alpha of high density lipoproteins purified from follicular fluid (FF-HDL). Further investigations demonstrated that sphingosine 1-phosphate (S1P), one of the lysophospholipids associated with HDL, accounts for the capacity of this lipoprotein to stimulate endothelial growth and the formation of new vessels. Activation of mitogen-activated protein kinase (p42/44(ERK1/2)), protein kinase C, and protein kinase Akt represent signaling pathways utilized by FF-HDL and S1P to induce endothelial proliferation and angiogenesis. We conclude that FF-HDL represents a novel mitogenic and angiogenic factor present in follicular fluid and that S1P is one of the FF-HDL lipid components accounting for this activity.     

Role of Activated Rac1/Cdc42 in Mediating Endothelial Cell Proliferation and Tumor Angiogenesis in Breast Cancer

Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.     

Inhibition of P53/miR‐34a improves diabetic endothelial dysfunction via activation of SIRT1

Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)‐treated endothelial cells (ECs) were subjected to pifithrin‐α (PFT‐α)—a specific inhibitor of P53, or P53‐small interfering RNA (siRNA), both of which attenuated the HG‐induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT‐α or P53‐siRNA prohibited P53 acetylation, decreased microRNA‐34a (miR‐34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR‐34a inhibitor (miR‐34a‐I) and PFT‐α increased SIRT1 protein level and alleviated the HG‐induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT‐α were completely abrogated by the miR‐34a mimic. In addition, SIRT1 inhibition by EX‐527 or Sirt1‐siRNA completely abolished miR‐34a‐I's protection against HG‐induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin‐induced diabetic mice, both PFT‐α and miR‐34a‐I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR‐34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR‐34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.     

PAKing up to the endothelium

Angiogenesis recapitulates the growth of blood vessels that progressively expand and remodel into a highly organized and stereotyped vascular network. During adulthood, endothelial cells that formed the vascular wall retain their plasticity and can be engaged in neo-vascularization in response to physiological stimuli, such as hypoxia, wound healing and tissue repair, ovarian cycle and pregnancy. In addition, numerous human diseases and pathological conditions are characterized by an excessive, uncontrolled and aberrant angiogenesis. The signalling pathways involving the small Rho GTPase, Rac and its downstream effector the p21-activated serine/threonine kinase (PAK) had recently emerged as pleiotropic modulators in these processes. Indeed, Rac and PAK were found to modulate endothelial cell biology, such as sprouting, migration, polarity, proliferation, lumen formation, and maturation. Elucidating the Rac/PAK molecular circuitry will provide essential information for the development of new therapeutic agents designed to normalize the blood vasculature in human diseases.     

The capacity of primordial follicles in fetal bovine ovaries to initiate growth in vitro develops during mid-gestation and is associated with meiotic arrest of oocytes

In cattle and other species in which the pool of resting, primordial follicles is formed during fetal life, little is known about the regulation of the early stages of ovarian follicular development. We used histological morphometry and a combination of observations in vivo and experiments in vitro to study the timing and regulation of follicle formation and the acquisition of the capacity of primordial follicles to initiate growth in cattle. In vivo, primordial, primary, and secondary follicles were first observed around Days 90, 140, and 210 of gestation, respectively. The long interval between the first appearance of primordial and primary follicles suggests that primordial follicles are not capable of activating when they are first formed, or they are inhibited from activating. This hypothesis was confirmed by the finding that most primordial follicles in pieces of ovarian cortex obtained from fetal ovaries older than 140 days activated (i.e., initiated growth) after 2 days in vitro, whereas follicles in cortical pieces from 90- to 140-day-old fetal ovaries did not. We tested the hypothesis that the oocytes of newly formed primordial follicles are not in meiotic arrest and found that before Day 141, most oocytes ( approximately 73%) were in prediplotene stages of prophase I, whereas after Day 140, the majority of oocytes ( approximately 85%) had arrested at the diplotene stage. This observation was further confirmed by the finding that levels of mRNA for YBX2, a protein associated with meiotic arrest, were 2.3 times higher in ovarian cortical pieces isolated after versus before Day 141. Primordial follicles in cortical pieces from 90- to 140-day-old fetal ovaries did activate during a longer, 10-day culture, but activation could be inhibited by adding estradiol or progesterone, but not dihydrotestosterone (all at 10(-6) M). Fetal ovaries secreted estradiol in vitro, and secretion by ovaries from 83 to 140-day-old fetuses declined precipitously ( approximately 30-fold) with age, consistent with the hypothesis that estradiol inhibits activation of newly formed primordial follicles in vivo. In summary, the results show that newly formed primordial follicles do not activate in vivo or within 2 days in vitro and that capacity to activate is correlated with achievement of meiotic arrest by the oocyte and can be inhibited by estradiol, which fetal ovaries actively produce around the time of follicle formation.     

Fibronectin promotes the elongation of microvessels during angiogenesis in vitro

Fibronectin is a component of the extracellular matrix of developing microvessels whose role in angiogensis is poorly understood. This study evaluated the effect of plasma fibronectin on angiogenesis in serum-free collagen gel culture of rat aorta. Aortic explants embedded in collagen gels generated microvascular outgrowths. Fibronectin incorporated in the collagen gel promoted a selective dose-dependent elongation of the newly formed microvessels without stimulating vascular proliferation. The fibronectin-treated microvessels were longer due to a proportional increase in the number of microvascular cells. However, fibronectin had no effect on microvascular DNA synthesis and mitotic activity. Fibronectin stimulated microvascular length also in cultures in which mitotic activity was suppressed and angiogenesis was markedly reduced by pretreating the aortic explants with mitomycin C. The synthetic peptide Gly-Arg-Asp-(GRGDS), which competes for the binding of fibronectin to its cell receptors and inhibits the adhesion of endothelial cells to substrates, arrested the elongation of developing microvessels causing regression and inhibition of angiogenesis. Conversely, Gly-Arg-Glu-(GRGES), which lacks the RGD sequence, had no inhibitory effect. These data support the hypothesis that fibronectin promotes angiogenesis and suggest that developing microvessels elongate in response to fibronectin as a result of an adhesion-dependent migratory recruitment of endothelial cells that does not require increased cell proliferation. © 1993 Wiley-Liss, Inc.     

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.     

Fibroblast growth factor 9 delivery during angiogenesis produces durable, vasoresponsive microvessels wrapped by smooth muscle cells

The therapeutic potential of angiogenic growth factors has not been realized. This may be because formation of endothelial sprouts is not followed by their muscularization into vasoreactive arteries. Using microarray expression analysis, we discovered that fibroblast growth factor 9 (FGF9) was highly upregulated as human vascular smooth muscle cells (SMCs) assemble into layered cords. FGF9 was not angiogenic when mixed with tissue implants or delivered to the ischemic mouse hind limb, but instead orchestrated wrapping of SMCs around neovessels. SMC wrapping in implants was driven by sonic hedgehog-mediated upregulation of PDGFRβ. Computed tomography microangiography and intravital microscopy revealed that microvessels formed in the presence of FGF9 had enhanced capacity to receive flow and were vasoreactive. Moreover, the vessels persisted beyond 1 year, remodeling into multilayered arteries paired with peripheral nerves. This mature physiological competency was attained by targeting mesenchymal cells rather than endothelial cells, a finding that could inform strategies for therapeutic angiogenesis and tissue engineering.     

Studying the growth of cervical carcinoma nests and angiogenesis by immunostaining, quantitation and three-dimensional structural analysis

OBJECTIVE: To analyze the relationship and mutual effect of the growth of cervical carcinoma nests and angiogenesis. STUDY DESIGN: Serial paraffin sections of cervical squamous carcinoma were stained in repeated order with hematoxylin-eosin (HE), immunostain for factor VIII-related antigen, type IV collagen and proliferating cell nuclear antigen (PCNA). Three-dimensional reconstructions were made, and the volumes of carcinoma nests, necrosis and microvessels were measured. RESULTS: Two types of cervical carcinoma nests were distinguished on the basis of their growth characteristics and vascularity (groups I and II). Group I nests: The carcinoma cells were proliferating actively, as determined by their morphology and PCNA staining. There were abundant microvessels. Some endothelial sprouts and cords penetrated the nests and then formed networks and new vessels. The volume ratio of microvessels, including sprouts and cords, to the nests was 0.6282:1. Group II nests: The center of these nests underwent necrosis. The peripheral cells were rather small, with no mitosis. The PCNA index was rather low; these nests grew very slowly. There were only a few surrounding microvessels with no endothelial sprouts or cords. The volume ratio of vessels to nest was 0.0657:1. CONCLUSION: Two types of cervical carcinoma nests show a close relationship and mutual effect of the growth of carcinoma nest and angiogenesis. Group I nests grow and develop well, with abundant microvessels. Thus, many tumor cells may be angiogenic and induce angiogenesis; growth of the nests seemed dependent on adequate neovascularization. Group II nests grow slowly, with a few microvessels; the center of the nests undergoes necrosis. The inadequate blood supply must be one of the important causes of necrosis. Considering that there must have been abundant neovascularization during their growth in the past, most of the microvessels must have been obliterated and then reabsorbed to make the remaining vessels so few.