The role of complement in ocular pathology

Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration.
Complement system
Complement has long been recognized as a critical component of the innate immune system [1–6]. It comprises of soluble and surface proteins that play a central role in host defense against infection and in the modulation of antigen-specific immune and inflammatory responses [4, 5]. Thus, complement also serves as a bridge between innate and adaptive immunity. The complement system can be activated by three proteolytic cascades namely, the classical, the alternative, and the lectin pathways (Fig. 1). Although the initial signal for the activation of each cascade differs, complement activation via these pathways triggers a sequence of biologic reactions in which each component is activated by the upstream component [3, 5]. This sequential activation of complement components by all three pathways leads to the formation of membrane attack complex (MAC). Under normal conditions, the activation of the complement system is kept under tight control by the coordinated action of soluble and membrane-associated complement regulatory proteins (CRegs). However, interference with the function and/or expression of CRegs leads to unwanted host tissue damage and tissue pathology due to unregulated complement activation [7, 8].
Fig. 1Complement system and complement regulatory proteins (CRegs). The complement system can be activated through three pathways and complement activation triggers a sequence of biological reactions which result in the generation of effector molecules (e.g., C3a, C3b, C5a, MAC) with potent immunological activities. The classical pathway can be activated by antigen-antibody complexes or by substances such a C-reactive protein. C1, C2, C4, and C3 are the components of the classical pathway. Activation of the alternative pathway does not require the presence of an antibody; C3, factor B, factor D, and properdin are the components of the alternative pathway. Like the alternative pathway, the activation of the lectin pathway is also antibody independent and can be achieved by interaction of serum lectins (e.g., mannose-binding protein, MBL), with sugar residues present on the bacterial cell wall. All three pathways converge on a common reaction that results in the formation of an enzyme—C3 convertase. This enzyme cleaves C3 into C3a and C3b. C3b forms C5 convertase that ultimately leads to the formation of membrane attack complex (MAC). The complement cascade is regulated at different check points (shown with black blocks) by CRegs. The soluble CRegs are shown in white boxes and the membrane-bound CRegs are shown in gray boxes
CRegs can be broadly categorized into two classes—membrane-bound CRegs and soluble CRegs (Fig. 1). Membrane-bound CRegs (Fig. 1) include decay-accelerating factor (DAF, CD55), membrane co-factor protein (MCP, CD46), complement receptor 1 (CR1, CD35), and membrane inhibitor of reactive lysis (MIRL, CD59). DAF, a glycosylphosphatidylinositol (GPI)-anchored protein prevents the activation of the complement system by inhibiting the formation of C3 and C5 convertases of both the classical and alternative pathways and accelerating the decay of these convertases [9, 10]. However, DAF does not irreversibly inactivate C3b and C4b and, after interaction with DAF, these molecules still possess cytolytic activity. MCP a transmembrane glycoprotein regulates the complement cascade by serving as a co-factor for serine protease factor I, which irreversibly inactivates the hemolytic potential of C3b and C4b [11, 12]. CR1 has been reported to possess both decay-accelerating and co-factor activities [13]. Like DAF, CD59 is also attached to the cell surface by GPI anchors. CD59 acts at the terminal step of complement activation and prevents the formation of membrane attack complex (MAC, C5b-9 complex), the final activation product of all three complement cascades by blocking the incorporation of C9 [14, 15]. Crry (5I2 antigen) is a C3 convertase inhibitor that possesses both decay-accelerating and membrane co-factor activities and present only in rodents [16, 17]. However, DAF, MCP, and CD59 have also been identified and characterized in rodents [18–23].
C1 inhibitor (C1INH), C4 binding protein (C4bp), complement factor H (CFH), complement factor I (CFI), S-protein and clustrin, are the examples of important soluble CRegs (Fig. 1). C1INH regulates activation of the classical complement pathway by inactivating the protease function of activated C1 complex. It also prevents spontaneous activation of C1 and binding to the zymogen forms of C1r and C1s [24]. C4bp acts as a co-factor for CFI, catalyzing the cleavage and inactivation of C4b [25]. CFH inhibits C3 activation by binding to C3b and acting as a co-factor for factor I-mediated cleavage of C3b and also has decay-accelerating activity for the alternative pathway C3 convertase, C3bBb. It also competes with factor B for surface-bound C3b [26]. Both clustrin and S-protein prevent the formation of MAC [6]. Soluble forms of MCP, DAF, CR1, CD59, and Crry have been reported to be present in various biological fluids in both humans and rodents [27–30]. Thus, a wide variety of CRegs (both membrane bound and soluble) have evolved to create efficient checkpoints at critical steps of complement cascades so that the host could be protected from the inadvertent activation of complement on its own tissue (Fig. 1).
Since complement is a powerful cytotoxic defense system, regulation of complement activation is crucial for host tissue damage control during inflammation especially in a sensitive organ like the eye. The eye is an immunologically privileged site and ocular immune privilege does not refer to the absence of immune system but, rather its fine regulation [31]. The studies discussed in this article highlight the role of complement activation and regulation in the protection of the normal eye. In addition, the studies discussed below demonstrate the importance of interplay between complement activation and complement regulation in the development of vision-threatening complications such as keratitis, uveitis, and macular degeneration.
Role of complement in the protection of the normal eye
It is important that we discuss the role of complement and CRegs in ocular protection under normal conditions before discussing their importance in ocular pathology. As mentioned above, the eye is an immune-privileged organ [31] and is highly vulnerable to various immunological insults. Therefore, it is not surprising that the complement system, which represents the first line of defense, plays an important part not only in protecting the eye from immunological insults but also in maintaining the immune-privileged state of the eye. Our results and several other independent studies have demonstrated the presence of various components of the complement system in the normal eye [2, 32–37]. We have further shown that ocular complement components are not just silent bystanders but are chronically active at a low level in normal eye [32]. We reported that both iC3b (activation product of C3) and MAC (end product of complement activation) are present in normal rat eye indicating that the complement system is continuously activated at a low level in the normal eye [32]. In 2003, we documented that the interaction between complement activation product iC3b and its receptor is vital for the creation of immunosuppressive environment that leads to the induction and the maintenance of ocular immune privilege and protection of intraocular structures that are critical for vision [38]. The presence and expression pattern of CRegs in the normal human eye was first described by us [33]. We reported that membrane-bound CRegs—MCP, DAF, and CD59—are differentially expressed in normal human and eyes [33]. Our studies along with other independent studies that were performed later demonstrated that Crry and CD59 (membrane bound and soluble) are also present in the normal rodent eye [32, 34, 35]. We have further shown that ocular CRegs are functionally active and tightly regulate the activation of intraocular complement [27, 32]. Functional inhibition of ocular CRegs using specific antibodies resulted in unregulated complement activation leading to severe intraocular inflammation in normal animals [32]. Furthermore, complement inhibitory activity in normal human intraocular fluid was blocked by inhibiting the function of MCP, DAF, and CD59 [27].
Thus, our results clearly established that within the normal eye the complement system seems to perform two important functions. First, the chronic low level of complement activation serves as a primary defense mechanism of the eye against pathogenic infection and is finely regulated by the soluble and membrane-bound intraocular complement regulatory proteins. This enables destruction of the putative pathogen without inadvertent damage of ocular tissue, which is vital for the maintenance of vision. Second, complement activation products (such as iC3b) generated as a result of the low level of complement activation results in the selective suppression of harmful T cell responses. In this role complement protects the eye from innocent bystander destruction associated with the T cell response to the invading pathogens. Complement components and CRegs present in normal tears have also been reported to be functionally active and have been proposed to serve as first line of defense in protecting the eye [36]. However, additional studies are required to determine the role of the complement system in normal tears.
Role of complement in corneal disease
The cornea is constantly exposed to various physical as well as immunological insults. Indeed the normal cornea is well equipped to protect itself from these insults and has been shown to possess functional complement activity [39–46]. In addition, low levels of C3, C3 split products, and MAC were detected in the normal cornea [32, 39, 46]. This observation further proves that low level of complement activation is taking place in the normal cornea since complement activation is necessary for the generation of these products. Studies reported in the literature suggest that complement cascade can be aggressively activated in the cornea during an immune and inflammatory reaction [45, 46]. Two reports by Mondino and colleagues described interesting sets of experiments [45, 46]. In these experiments, the normal cornea from human donors was exposed to lipopolysaccharide (LPS), ribitol teichoic acid immune complex, acid (HCl), or alkali (NaOH) separately and the generation of anaphylatoxins—C3a, C4a, and C5a—as well as MAC in the corneas was monitored. It was reported that anaphylatoxins—C3a, C4a, and C5a—could be generated when the cornea was injured with LPS, immune complexes, acid, or alkali. Interestingly MAC could only be generated when the cornea was exposed to LPS or immune complexes. Cornea failed to generate MAC when insulted with acid or alkali. The immune response mounted to LPS or immune complex is similar to that generated against infectious agents like gram-negative bacteria. Indeed the complement system has been shown to play a critical role in protection against Pseudomonas aeruginosa infection that causes keratitis [47, 48]. Additionally, complement activation is believed to play an important role in ulceration of human cornea induced by gram-negative bacteria [49].
The aforementioned observations have important clinical implications. If in future anti-complement agents were to be considered for the treatment of corneal pathology, anaphylatoxins would be an ideal target for the patient presenting with chemical injury such as exposure to acid or alkali. On the other hand, in the case of bacterial infection both anti-anaphylatoxin and anti-MAC therapy should be considered. Although as discussed below the cornea possesses the ability to express different CRegs to protect itself from complement-mediated damage, it may not be able to upregulate various CRegs to an appropriate level during an acute episode of complement activation as observed during chemical insults or acute bacterial infection. In such cases, topical application of recombinant CRegs could be beneficial and serve as a better alternative for intervention.
Enhanced complement activation can cause damage to the autologous corneal tissue during an infection or inflammation. Membrane-bound CRegs such as MCP, DAF, Crry (Fig. 2a), and CD59 (Fig. 2b) are expressed in the different layers of the cornea to protect this tissue from complement-mediated damage [27, 33, 50]. Interestingly, compared to corneal stroma and corneal endothelium, various CRegs are very strongly expressed on the corneal epithelium [33] (Fig. 2a and b). This high expression of CRegs on the corneal epithelium is crucial for the protection of the cornea because the cornea is continually exposed to various pathogens including bacteria. These bacteria produce phospholipase and other enzymes which can remove GPI-anchored DAF and CD59 from the corneal surface [51]. Thus, in the absence of a very high expression of CRegs on the corneal epithelium, bacterially induced loss of DAF and CD59 could be deleterious to the cornea and lead to vision loss after the putative pathogen has been destroyed by the complement system. To investigate if a deficiency or abnormality in the expression of CRegs may play a role in ocular surface disease, systemic examination of corneal tissue from patients with different diseases such as pseudophakic bullous keratopathy, HSV-1 keratitis, and herpes zoster scleritis is needed.
Fig. 2Complement regulatory proteins in normal cornea. The figure shows immunofluorescent staining for Crry (a) and CD59 (b) in the cornea of naive Lewis rat. Objective magnification ×20. Epi Epithelium, S stroma, Endo endothelium, AC anterior chamber
Role of complement in autoimmune uveitis
Uveitis, the inflammation of the uveal tract, can be classified anatomically as anterior, intermediate, posterior, or diffuse (panuveitis) depending on the segment of the eye that is affected. Each year, approximately 17% of active uveitis patients experience some degree of vision loss [52]. Uveitis may be idiopathic, associated with systemic diseases such as Behçet’s disease, and Vogt–Koyanagi–Harada disease or resulting from a variety of infectious agents. Inflammation resulting from uveitis may lead to conditions like cataract, glaucoma, and cystoid macular edema that may cause irreversible vision loss. Anterior uveitis is the most common form of uveitis, and the most common form of anterior uveitis is of unknown (i.e., idiopathic) etiology [52].
Although the exact cause of the disease is still unknown, a wide array of studies using different animal models have established that the inflammation in uveitis is due to an autoimmune response to various ocular antigens [53–56]. Unfortunately, almost all of the studies in the past three decades have focused on the role of T cells in the pathogenesis of uveitis [54, 57, 58]. Very few laboratories have investigated the role of the complement system in the development of autoimmune uveitis [32, 58–66].
Through our recent studies, the critical role of complement and complement regulatory proteins (CRegs) in the pathogenesis of idiopathic anterior uveitis was addressed and recognized. Our laboratory has used experimental autoimmune anterior uveitis (EAAU)—an animal model of human autoimmune anterior uveitis—to investigate the role of the complement system, a significant and previously neglected facet of uveitis [53, 62, 63]. EAAU is induced by injecting bovine melanin-associated antigen emulsified in Freund’s complete adjuvant, in the foot pad of Lewis rats [53]. The onset of the disease (days 12–14 post immunization) is marked by inflammation of the iris/ciliary body followed by massive infiltration of the inflammatory cells in the anterior segment of the eye during the peak (days 16–19 post immunization) of EAAU (Fig. 3). The inflammation resolves after approximately 3 weeks, and the eye returns to the normal state with no apparent sign of any tissue damage (Fig. 3). Interestingly, studies from our laboratory have demonstrated that the activation of the complement system is critical for the development of EAAU [62]. During EAAU, the complement system is activated (detected by Western blot analysis for iC3b) in the eye, and the kinetics of complement activation follows a pattern that is similar to the clinical course of the disease. Furthermore, depletion of the complement system of the host resulted in complete inhibition of EAAU. In complement-depleted rats, the levels of IFN-γ, IP-10, ICAM-1, and LECAM-1 were extremely low compared to the complement-sufficient rats during EAAU. Our observations suggest that the activation of the local complement system plays a critical role in the development of inflammation during EAAU and suppression of intraocular complement system, thus may provide a successful strategy for uveitis therapy. At present, the exact mechanism and pathways involved in local complement activation during uveitis are not known. It is also not known if the increase in complement components in the anterior segment of the eye is due to the upregulation of their synthesis by resident ocular cells or is a result of spillover from the systemic complement system. Previous studies from our laboratory established that a functionally active complement system is present in the anterior segment of the eye because when a well-known activator of complement such as zymosan was injected in the anterior chamber, severe anterior uveitis was induced. This zymosan-induced uveitis was due to the activation of the complement system because anterior uveitis was completely inhibited when the complement system of the host was depleted [32]. Furthermore, we and others have reported the presence of various complement components in aqueous humor as well as other parts of the normal eye [32, 39, 41, 59, 61]. These observations taken together suggest that ocular complement is activated during the course of EAAU and the inhibition of the intraocular complement system may lead to more effective management and/or treatment of autoimmune uveitis.
Fig. 3Clinical and histologic pictures of rat eye during different stages of experimental autoimmune anterior uveitis (EAAU). No inflammation could be detected in the iris (I) and the ciliary body (CB) after clinical (a) and histologic (d) examination at day 8 post immunization (before the onset of EAAU). At the peak of disease (day 19 post immunization) the anterior chamber (AC) appeared cloudy and opaque due to the presence of proteinaceous material and inflammatory cells (b). Histopathologic picture of rat eye at the peak of EAAU (e) revealed severe inflammation of the iris (I) and ciliary body (CB). Heavy infiltration in the anterior chamber (AC) with spillover into the anterior vitreous was also observed at this time point. The eye appeared normal both clinically (c) and histologically (f) after the resolution of EAAU (day 30 post immunization). Objective magnification ×10
When considering anti-complement therapy, we cannot ignore the role of CRegs in the resolution of autoimmune uveitis. We have demonstrated that the functionally active CRegs downregulate intraocular complement activation and are critical to protect the ocular tissues from complement-mediated damage during EAAU [63]. We observed that various CRegs were upregulated during the course of EAAU on the resident ocular tissue and this upregulation was not due to the infiltrating inflammatory cells because the levels of CRegs remained upregulated after the resolution of the disease when all the infiltrating cells were cleared from the anterior segment of the eye [63]. Furthermore, when the function or expression of CRegs was inhibited in vivo, more severe EAAU with early onset and delayed resolution was observed. These observations demonstrate that ocular tissue can protect itself by upregulating various CRegs during uveitis and provide strong evidence for the use of recombinant CRegs as therapeutic agents in the treatment of autoimmune uveitis.
Activation of the complement system has also been reported in other animal models of anterior uveitis such as endotoxin-induced uveitis (EIU) [64, 65]. In EIU, it was demonstrated that the complement system is critical for the development of inflammation, and there is a local activation of the complement system as evident by accumulation of complement components (C1q, C3, and C4) in various parts of the eye during inflammation [66]. Activation of the complement system was also implicated in experimental allergic uveitis [67]. Using experimental autoimmune uveoretinitis (EAU) animal model, the complement system has been reported to play an important role in the pathogenesis posterior uveitis [68]. Recently, Read and co-workers demonstrated that the transgenic mice that express soluble Crry in the retina had decreased incidence and severity of EAU [68]. In these transgenic mice, EAU was not completely suppressed. This may be due to relatively low levels of Crry expressed compared with the levels that are required for the complete inhibition of the complement system. It is possible that a higher dose of exogenous Crry or similar CReg may result in complete inhibition of EAU.
Thus, these studies provide strong evidence that the complement system and CRegs play an important role in the pathogenesis of autoimmune uveitis and provide alternative approaches for the development of effective therapy. More research in the future will present us enough information to develop anti-complement therapy for uveitis. Hopefully, in the future, complement inhibitors might be used as novel anti-uveitic agents in the clinic for the treatment of this important form of human ocular disease.
Role of complement in age-related macular degeneration
Age-related macular degeneration (AMD) is the most common cause of legal blindness worldwide among the elderly over the age of 50. Approximately 1.8 million people in the United States alone have AMD and it is projected that by 2020, roughly 2.9 million people will develop this disease [69–71]. In AMD, there is a progressive destruction of the macula leading to the loss of central vision. AMD is classified into non-exudative (dry type) and exudative (wet type) [69–71]. In the dry type of AMD, drusen—small yellow deposits—are formed between the retinal-pigmented epithelium (RPE) and Bruch’s membrane [72]. In wet-type AMD, abnormal vessel growth takes place from choroid, termed choroidal neovascularization (CNV), under the retinal pigment epithelium [71, 72]. Although several risk factors are associated with AMD, the exact pathogenesis still remains unknown. The last few years have witnessed an amazing series of research establishing the central role of the complement system in the pathogenesis of AMD both in humans and in experimental animals. Various studies in the literature have indicated a potential role for complement in drusen formation in the non-exudative form of AMD in humans [73–75]. Complement components, complement activation products (C3a, C5a, MAC), and complement regulatory proteins (CD46, Vitronectin) have been localized in drusen in patients with AMD [73–75].
The discovery of allelic variants of complement factor H (CFH) as a major risk factor for AMD has become a landmark study [76–79]. The polymorphism that gives rise to this allelic variant of CFH results from a tyrosine to histidine replacement at position 402. The “high risk” variant, i.e., homozygous for histidine residue at position 402 is associated with five- to sevenfold increased risk of AMD [76–79]. CFH consists of 20 short consensus repeats (SCRs) and the polymorphic site 402 resides in SCR7. SCR7 contains the binding site for C3b, glycosaminoglycans (GAG) and C-reactive protein (CRP). The polymorphism at position 402 in SCR7 reduces the binding affinity to CRP [80] and GAG [81]. Binding of CFH to CRP and GAG plays an important role in the regulation of the alternative pathway. Thus the polymorphic form of CFH with histidine at position 402 may have reduced ability to regulate the activation of the alternative pathway. This may lead to uncontrolled activation of alternative pathway which may result in the development of AMD. The loss of regulatory activity of CFH due to polymorphism can have a direct effect on the predisposition of the eye to AMD as it has been shown recently that CFH is expressed locally in the optic nerve, retina, and retinal pigment epithelium [82]. The importance of CFH in AMD was further demonstrated in a recent study. It was reported that the aged mice genetically deficient in CFH (cfh−/−) develop spontaneous changes in retina (e.g., photoreceptor dysfunction, increased deposition of C3 in retina, thinning of Bruch’s membrane) which is consistent with those seen in AMD [83].
Polymorphic variants of other complement components have also been shown to be associated with AMD. In a recent study, Gold et al. reported that complement components—factor B (CFB) and complement component 2 (C2)—are also associated with AMD [84]. In this report, the investigators identified high risk allele and two protective alleles for CFB and C2 that were associated with AMD. An independent study further established a similar association between CFB, C2, and AMD [85]. More recently, investigators compared 847 patients with AMD with 701 unaffected people and observed that a variant in the complement C3 gene affected the risk of developing AMD [86].
The aforementioned studies establish that the polymorphism in the genes of certain complement components and regulators genetically predisposes some individuals to AMD. However, we should also pay attention to the fact that other risk factors for AMD such as alcohol consumption and smoking nicotine can directly affect the level of complement activation and CRegs. For instance, chronic ethanol consumption upregulates CFB but downregulates CFH in mice [87]. Thus, association of alcohol consumption with AMD may be due to its direct effect on the complement system. Indeed we have reported that chronic alcohol consumption increases the size of CNV complex in rat model of laser-induced CNV [88]. Similarly, cigarette smoking has been shown to decrease the affinity of CFH for C3 and activate the complement system [89].
In our laboratory, we use a mouse model of laser-induced CNV to study the role of complement in the pathogenesis of wet-type AMD. A reliable way to produce CNV in animals is to rupture Bruch’s membrane with laser photocoagulation [90–92]. We and others have found that choroidal neovascularization induced in rodents by laser photocoagulation is useful to gain insights into the pathogenesis of new vessel growth from the choroid [90–96]. In 2005, we demonstrated for the first time using this animal model that complement activation and generation of MAC is critical for the development of laser-induced CNV. In this study, C3−/− mice and anti-C6-treated mice failed to develop CNV [93]. As mentioned above, the importance of C3 in the pathogenesis of AMD was recently confirmed by Yates et al., who established an association of C3 polymorphism with AMD [86]. We further observed that in the absence of MAC deposition, release of angiogenic factors such as vascular endothelial growth factor (VEGF), TGF-β2, and β-FGF was inhibited. This is a crucial observation as it suggests that the complement system is a potential therapeutic target for the treatment of CNV. Subsequently, we reported that the activation of complement by alternative pathway is responsible for MAC formation in laser-induced CNV. This alternative pathway activation was due to the decrease in CFH levels and increase in CFB levels in the RPE/choroid after laser treatment [94]. In a separate study, we used recombinant-soluble CD59-Fc chimeric protein to inhibit the formation of MAC after laser photocoagulation [95]. Treatment of mice with recombinant-soluble CD59-Fc inhibited the development of choroidal angiogenesis in the laser-induced mouse model by blocking MAC formation which in turn blocked the induction/release of angiogenic growth factors such as VEGF, β-FGF, and TGF-β2 (Fig. 4). Thus, our studies suggest that the recombinant forms of CD59 could be used as a potential drug in the future to treat CNV. Another potential target could be anaphylatoxins—C3a and C5a. It was reported that C3a and C5a are present not only in human drusen but are also localized in the choroid of mice with laser-induced CNV [97].
Fig. 4Role of CD59 in laser-induced choroidal neovascularization (CNV). Laser photocoagulation results in complement activation and MAC formation in the posterior segment of the eye. MAC is a complex assembly of C5b to C9 components with multimers of C9 forming a pore in the cell membrane. This causes increased release of growth factors—VEGF, TGFβ2, and β-FGF—which in turn leads to the development of CNV. The membrane-bound as well as recombinant-soluble (rs) CD59, bind to C9 and C8 and inhibit the polymerization of C9 which is prerequisite for MAC formation. In the absence of MAC, release of angiogenic factors is downregulated. This inhibits the growth of new vessel from the choroid (CNV)
Using the mouse model of laser-induced CNV, we observed that the CH50 levels in the serum of lasered mice varied when compared to non-lasered mice [93, 94]. Another study demonstrated that the plasma complement C3a des Arg levels increased in AMD patients compared to controls [98]. This increase in plasma C3a des Arg concentration reflects systemic complement activation. Thus systemic activation of the complement system may also contribute to the pathogenesis of AMD.
Currently, several different approaches are being used to treat wet AMD in humans [99]. Unfortunately, these treatment options have limitations due to their short-term and serious side effects. Although, photodynamic therapy (PDT) reduces the rate of vision loss in most patients, it does not lead to significant improvement in vision. Furthermore, repeated PDT can cause severe damage to the posterior segment of the eye and is not cost effective. Two drugs Lucentis and Macugen are approved by Federal Drug Administration for the treatment of AMD and both drugs inhibit vascular endothelial growth factor. One of the major drawbacks of Lucentis or Macugen is that repeated injections of these drugs are needed to treat AMD. Repeated injections can cause some serious side effects including hemorrhage of the eye membrane, eye pain, and vitreous floaters. Therefore, alternative therapeutic strategies with minimum side effects are required to better treat AMD patients. We believe that the results derived from the studies reviewed above would open up the window of opportunities for the development of anti-complement therapy in the treatment of AMD.
In view of important developments relative to the role of the complement system in AMD during the past several years, we believe that the next decade would be critical for such studies. We hope that the interest will continue to grow in the future so that an alternative therapy could be developed by inhibiting complement activation. Since untreated CNV leads to the irreversible loss of central vision, it is important for the pharmaceutical companies to utilize the knowledge available to them from different research laboratories and should make every effort to transfer this knowledge to develop new drugs and therapies for AMD patients.
Conclusions and clinical implications
During the last decade, a lot of progress has been made to understand the pathophysiology of corneal diseases, uveitis, and macular degeneration. Specifically, the studies defining the role of the complement system in ocular diseases have drawn a lot of attention. It is an exciting area for future investigations and the basic questions such as what triggers the complement activation and which specific pathway is involved in corneal diseases, uveitis and macular degeneration need to be addressed. This understanding will help us to develop better therapies for ocular diseases in the future so that blindness could be prevented.
The evidence derived from the studies reviewed here supports the concept that recombinant complement regulatory proteins may be therapeutically useful in the prevention of ocular tissue damage and in the treatment of various ocular diseases. Agents that specifically inhibit the complement system or complement activation products have been proven beneficial in the treatment of both human and experimental diseases over the past few years. Thus, the therapy based on complement inhibition has great potential in the future for the treatment of various ocular diseases such as keratitis, uveitis, and macular degeneration.

complement system
complement regulatory proteins
eye
uveitis
age-related macular degeneration
corneal inflammation


Evaluation of left atrial systolic function in noncompaction cardiomyopathy by real-time three-dimensional echocardiography

Background Noncompaction cardiomyopathy (NCCM) is a rare disorder with persistance of the embryonic pattern of myoarchitecture. NCCM is characterized by loosened, spongy myocardium associated with a high incidence of systolic and diastolic left ventricular (LV) dysfunction and heart failure (HF). It is known that LV dysfunction contributes to elevated left atrial (LA) and pulmonary vascular pressures, however atrial function has not been examined in NCCM. The objective of the present study was to assess LA systolic function characterized by LA ejection force (LAEF) in NCCM patients using real-time three-dimensional echocardiography (RT3DE) and to compare to control subjects. Methods The study comprised 17 patients with an established diagnosis of NCCM and their results were compared to 17 healthy age-matched controls with no evidence of cardiovascular disease. Forty-one percent of NCCM patients were in NYHA functional class II / III HF. Previously proposed echocardiographic diagnostic criteria for NCCM were used. All patients underwent conventional two-dimensional echocardiography and RT3DE. LAEF was measured based on MA annulus diameter (LAEF3D-MAD) and area (LAEF3D-MAA) using RT3DE. Results The presence and severity of mitral regurgitation were more frequent in NCCM patients than in control subjects. LV diameters and mitral annulus were significantly increased in NCCM patients. Compared with control subjects, both LAEF3D-MAD (3.8 ± 2.2 vs 2.3 ± 1.0 kdyne, P < 0.05) and LAEF3D-MAA (12.7 ± 7.6 vs 4.9 ± 2.1 kdyne, P < 0.01) were significantly increased in NCCM patients. Conclusions LAEF as a characteristic of LA systolic function is increased in NCCM patients compared to normal individuals. These results can suggest compensating left atrial work against the dysfunctional LV in NCCM patients.
Introduction
Noncompaction cardiomyopathy (NCCM) is a rare disorder with persistance of the embryonic pattern of myoarchitecture, and has been assumed to occur as a consequence to intrauterine arrest of compaction [1]. NCCM is characterized by loosened, spongy myocardium associated with a high incidence of systolic and diastolic left ventricular (LV) dysfunction and heart failure. It is known that LV dysfunction contributes to elevated left atrial (LA) and pulmonary vascular pressures, however atrial function has not been examined in NCCM. It is difficult to assess non-invasively LA contraction, but Manning et al. reported a non-invasive method for the evaluation of LA ejection force (LAEF) as a characteristic of LA systolic function [2]. Anwar et al. demonstrated the usefulness of real-time three-dimensional echocardiography (RT3DE) for the evaluation of LAEF [3]. The objective of the present study was to assess LAEF in NCCM patients using RT3DE and to compare to control subjects.
Methods
Patient populations
The study comprised 17 patients with an established diagnosis of NCCM and their results were compared to 17 healthy age-matched controls with no evidence of cardiovascular disease. All patients and control subjects were in sinus rhytm and all of them were examined by two-dimensional echocardiography (2DE) and RT3DE.
Diagnostic criteria for NCCM
Previously proposed echocardiographic diagnostic criteria for NCCM by Jenni et al. were used to include patients into the present study from the Outpatient Clinic of Erasmus Medical Center, Rotterdam during 3 years [4]: (1) absence of coexisting cardiac anomalies, (2) segmental, excessive thickening of the LV wall with a two-layered structure: a thin, compacted epicardial layer and a much thicker, noncompacted layer with the characteristic appearance of numerous, prominent trabeculations (meshwork) and deep intertrabecular recesses, (3) colour Doppler evidence of deeply perfused intertrabecular recesses, and (4) predominant localization of thickening in the LV apical, midlateral, and midinferior walls. Hypertensive heart disease was excluded by clinical and echocardiographic examinations (septal thickness < 13 mm).
2DE examination
The 2DE was undertaken with the patient lying in the left lateral decubitus position using both apical and parasternal views. The 2DE studies were performed using a 3.5-MHz transducer and a commercially available ultrasound system (Sonos 7500, Philips, Best, The Netherlands). The following measures were obtained: (1) LV end-diastolic diameter (LV-EDD) and LV end-systolic diameter (LV-ESD) by M-mode echocardiography; (2) LV fractional shortening (LV-FS); (3) peak velocities of transmitral E and A waves with pulsed Doppler and (4) mitral regurgitation graded as mild (jet area < 4 cm2), moderate (jet area 4–8 cm2), and severe (jet area > 8 cm2) according to maximum jet area by color Doppler.
RT3DE examination
RT3DE was performed using the same ultrasound system, and performed with a X-4 matrix transducer capable of providing real-time B-mode and colour Doppler. The 3D images were collected within 5–7 s of breath holding in full-volume mode. The 3D data were transferred to an offline analysis system (TomTec Inc., Munich, Germany). Data were stored digitally and subsequently evaluated by two echocardiographers. Data analysis of RT3DE imaging was based on a two-dimensional approach relying on the echocardiographic images obtained from the apical views and on manual tracing of inner border of the mitral annulus (MA). Once this was completed the surface area was automatically delineated and could be visualized from different points of views. Manual modification was done to correct any image if necessary. The following measures were obtained: (1) MA diameter (MAD3D) and area (MAA3D) measured at end-diastole (just before mitral valve closure) and (2) LAEF3D-MAD and LAEF3D-MAA calculated by formula as 0.5 × 1.06 × (MAD3D or MAA3D) × V2, where V is the peak velocity of A wave, 1.06 gm/cm3 is blood viscosity and 0.5 is a coefficient factor [2] (Fig. 1).
Fig. 1Automatically cropped two-dimensional image from real-time three-dimensional echocardiographic dataset demonstrating left ventricular cavity in a patient with noncompaction cardiomyopathy (A). Measurement of mitral annulus area and mitral inflow A wave velocity can help calculation of left atrial ejection force as a characteristic of atrial systole. Real-time three-dimensional echocardiography allows the `en-face` evaluation of mitral annulus helping exact measurement of mitral annulus diameter and area (B). Abbreviations: MA: mitral annulus, MAD: mitral annulus diameter, E and A: mitral inflow E and A wave velocity measured by pulsed Doppler
Statistical analysis
All data obtained by 2DE or RT3DE were presented as mean ± SD. Data analysis was performed using statistical software (SPSS, Version 12.1, SPSS Inc, Chicago, USA). Independent sample t test was performed to compare between means of variables of groups to determine the statistical significance of difference. The level of significance was set to p < 0.05. In our department, an excellent correlation (r = 0.82, P < 0.0001) has been described between MAD3D and MRI measurements of MAD [5]. There was no significant difference between MAD3D and MADMRI. Similarly, there was no difference between MAA3D and MAAMRI.
Results
Clinical and 2D echocardiographic data
Clinical and echocardiographic data of NCCM patients and controls are shown in Tables 1 and 2. The presence and severity of mitral regurgitation were more frequent in NCCM patients than in control subjects. LV diameters were significantly increased, while LV-FS was significantly decreased in NCCM patients. The number of noncompacted segments was 5.4 ± 2.9 in NCCM patients, which did not show any correlation with any echocardiographic parameters.
Table 1Demographic and clinical dataParameterNCCM patients (n = 17)Controls (n = 17)Age (years)48 ± 2043 ± 10Male sex (%)8 (47)11 (65)Diabetes mellitus (%)9 (53)10 (59)Hypercholesterolaemia (%)10 (59)11 (65)NYHA-class II/III HF (%)3 (18)/4 (24)0 (0)Abbreviations: NCCM: noncompaction cardiomyopathy, NYHA-class II/III HF: New York Heart Association functional class II/III heart failureTable 2Echocardiographic dataParameterNCCM patients (n = 17)Controls (n = 17)2D echocardiography    LV-EDD (mm)60.6 ± 11.0 **51.2 ± 6.6    LV-ESD (mm)48.1 ± 13.0 **33.0 ± 6.7    LV-FS (%)22.1 ± 17.1 **35.7 ± 6.4    Mitral inflow A wave velocity (cm/s)50.1 ± 16.2 *41.8 ± 9.2Mitral regurgitation    No8 (47) *15 (88)    Mild6 (35) *2 (12)     Moderate-severe3 (18) *0 (0)RT3DE examination    MAD3D (cm)3.5 ± 0.5 *3.2 ± 0.3    MAA3D (cm2)12.0 ± 2.8 *7.9 ± 1.4    LA-EF3D-MAD (kdyne)3.8 ± 2.2 *2.3 ± 1.0    LA-EF3D-MAA (kdyne)12.7 ± 7.6 **4.9 ± 2.1* P < 0.05 vs controls**P < 0.01 vs controlsAbbreviations: 2D: two-dimensional, 3D: three-dimensional, A: mitral inflow A wave velocity measured by pulsed Doppler, D: diastolic, EDD: enddiastolic diameter, ESD: endsystolic diameter, FS: fractional shortening, NYHA-class II/III HF: New York Heart Association functional class II/III heart failure, LA-EF: left atrial ejection force, LV: left ventricular, MAA: mitral annulus area, MAD: mitral annulus diameter, NCCM: noncompaction cardiomyopathy
RT3DE data
Acquisition and postprocessing of RT3DE data were performed successfully with clear delineation of the MA in all patients. Compared with control subjects, MAD3D and MAA3D were significantly larger, while LAEF3D-MAD and LAEF3D-MAA were significantly increased in NCCM patients (P < 0.05) (Table 2).
Discussion
Left atrial ejection force (LAEF), based on classic Newtonian mechanics, provides a physiological assessment of atrial systolic function and is a potentially useful index for assessing atrial contribution to diastolic performance. To the best of authors` knowledge this is the first time to demonstrate increased LAEF during RT3DE indicating high LA workload in NCCM patients. NCCM is known to be a relatively new clinicopathologic entity characterized by a pattern of prominent trabecular meshwork and deep intertrabecular recesses communicating with the LV cavity [1]. Results of recent studies confirmed the hypothesis that isolated ventricular noncompaction can be a part of a more widespread cardiomyopathy, involving both the morphologically normal and dysmorphic LV myocardial segments [6–9]. However, the pathophysiological mechanisms of LV dysfunction remained unclear and, moreover little is known about LA function in NCCM.
Manning et al. surmised that LAEF is equal to left ejection volume times acceleration of LA flow according to Newton’s law of motion (force equals mass times acceleration) [2]. Anwar et al. confirmed that RT3DE is a useful method for the evaluation of LAEF3D-MAA [3]. In recent studies MAA3D has been confirmed to be traced successfully with good interobserver agreement and comparable to magnetic resonance imaging [5]. RT3DE allowed exact ‘en-face’ measurement of MAA3D suggesting overstretching and dilatation in dilated [10], hypertrophic [3, 10] and noncompaction cardiomyopathy (present study). LAEF was higher in hypertrophic cardiomyopathy (HCM) patients compared with control subjects, with the highest value found in patients with obstructive HCM due to higher LV resistance [3]. LAEF3D-MAA calculated by the MAA3D-derived formula was higher in HCM patients than by the ‘mitral valve area’-derived formula either by 2D echocardiography or RT3DE [3].
In the present study, 41% of NCCM patients were in NYHA functional class II / III HF. Recently, Triposkiadis et al. demonstrated that in patients with HF, LAEF and LA kinetic energy are higher than in normal controls [11]. This is most likely due to LA dilation, which compensates for the depressed LA systolic shortening and increases LA active emptying volume and LA contribution to LV stroke volume, maintaining LV stroke volume and cardiac output [11, 12]. However, it should be noted that other authors found decreased LA kinetic energy in patients with advanced HF (NYHA functional class III HF) [13, 14]. It has been concluded that LA systolic failure contributes to the transition from moderate to advanced HF.
Limitations
During evaluation of LA systolic function, consideration of other factors affecting LAEF is important. According to recent findings, age considerably influences LAEF [15]. This fact could also affect our results, but no significant differences were found in age between NCCM patients and controls. LA systolic function is deteriorated in patients with sick sinus syndrome and in those with paroxysmal atrial fibrillation [16]. However, all of our patients were in sinus rhythm. Cioffi et al. demonstrated that LV geometry can also influence LA size and function [17]. The concentric LV pattern was associated with the greatest LA volumes and with the strongest LAEF, suggesting that chronic pressure overload more than volume overload stimulates the LA in increasing its workload. Left atrial dilation can be resulted from chronic mitral regurgitation, which can also affect LA systolic function. Another important limitation is that left atrial contraction produces both the forward blood flow toward the LV through the mitral valve and backward flow towards the pulmonary veins. Unfortunately, only the forward blood flow was examined during the present study.
Conclusions
LAEF is increased in NCCM patients compared to normal individuals. These results can suggest compensating left atrial work against the dysfunctional LV in NCCM patients. However, further studies are warranted evaluating LA function by RT3DE in patients with different stages of NCCM.

noncompaction
three-dimensional echocardiography
atrial function


Mixed-type gastric carcinomas exhibit more aggressive features and indicate the histogenesis of carcinomas

To investigate the pathobiological behaviors of gastric mixed-type (MT) carcinomas and gastric carcinogenesis, the clinicopathological characteristics of MT carcinomas were analyzed and compared with intestinal-type (IT) and diffuse-type (DT) carcinomas. The expression of Ki-67, caspase-3, p53, fragile histine triad (FHIT), maspin, extracellular matrix metalloproteinase inducer (EMMPRIN), vascular growth factor (VEGF), MUC-2, 4, 5AC and 6, CD44, E-cadherin, β-catenin, and phosphorylated glycogen synthase kinase 3β-ser9 (P-GSK3β-ser9) was examined on tissue microarrays using immunohistochemistry. It was found that MT carcinomas exhibited large size, deep invasion, frequent local invasion, and lymph node metastasis in comparison with IT and DT carcinomas (p < 0.05). All the markers except MUC-5AC showed higher expression in IT than DT carcinomas (p < 0.05). The expression of maspin, EMMPRIN, VEGF, MUC-4, and membrane E-cadherin was stronger in MT intestinal than diffuse component (p < 0.05). Immunoreactivities to Ki-67, EMMPRIN, and VEGF were weaker in IT carcinoma than in the MT intestinal portion (p < 0.05), while the opposite was true for CD44, MUC-2, and MUC-6 (p < 0.05). The MT diffuse component displayed a higher expression of FHIT, VEGF, and P-GSK3β-ser9 than DT carcinoma (p < 0.05). The accumulative survival rate of the IT carcinoma patients was higher than the other types (p < 0.05). The invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent prognostic factors for gastric carcinomas (p < 0.05). These findings suggested that MT carcinomas were also indicated to be more aggressive than IT and DT carcinomas. Significant differences were observed in the proliferation, apoptosis, angiogenesis, mucin secretion, and cell adhesion between IT and DT carcinomas, whereas only a few of these characteristics showed differences between the MT intestinal and diffuse parts, thus suggesting that both the MT components might originate from the stem cells with similar genetic traits, but follow different histogenic pathways.
Introduction
Gastric carcinoma is the second leading cause of cancer-related death behind lung carcinoma despite a worldwide decline in both its incidence and mortality since the late half of the twentieth century [11]. It continues to be a major health problem because of the slow decrease in incidence in Asia and high mortality of diagnosed gastric carcinoma in the West [23]. Generally, the prognosis of patients with gastric carcinoma is somewhat dependent on its histological type, such as intestinal type (IT) or diffuse type (DT) as defined by Lauren [16].
Although gastric carcinoma is a malignant tumor originating from the same gastric epithelium, its morphological features vary substantially among individual patients. Histologically, IT carcinoma principally includes papillary, well-differentiated, and moderately-differentiated or mucinous adenocarcinoma without signet ring carcinoma (SRC) cells, whereas DT mainly consists of the SRC, poorly differentiated and undifferentiated adenocarcinoma of WHO classification [7, 23]. Although Kushima and Hattori [12] firstly proposed an entity of gastric-type carcinoma and terminologically seemed homologous to the IT one, the carcinoma cells of this type closely resemble normal foveolar cells on the basis of tissue morphology and mucin properties and are considered to have been derived from foveolar hyperplasia or pyloric gland adenoma [12, 13]. Recently, our group also focused on Lauren's classification and found that IT carcinoma frequently occurred in old men, while DT was more frequent in comparatively young women. The latter group was more inclined to invasion into the muscularis propria, lymphatic invasion, and lymph node metastasis, and belonged to a higher TNM staging, in comparison to its IT counterparts. Further analysis demonstrated that IT gastric carcinomas with a more favorable prognosis were prone to high levels of proliferation and apoptosis and also always accompanied by a strong expression of the fragile histine triad (FHIT), phosphatase and tensin homology deleted from human chromosome 10 (PTEN), p53, and extracellular matrix metalloproteinase inducer (EMMPRIN) [30]. Additionally, many genetic and epigenetic changes, such as mutation, deletion, loss of heterozygosity, methylation, and microsatellite instability were differentially observed in both histological types of gastric carcinoma [27, 36]. These data indicate that different carcinogenic pathways exist for gastric IT and DT carcinomas. Although carcinogenesis is a multistage process which consists of a multi-factorial etiology resulting from gene–environment interaction, the intestinal type is frequently related to environmental factors like diet and predominates in areas with a high incidence of this disease. In contrast, the diffuse type is thought to be of genetic origin and evenly distributed worldwide [30, 24]. It is commonly believed that Lauren's classification is valuable for both the epidemiological studies and gastric carcinogenesis.
However, a small number of gastric carcinomas remain characterized as unclassified or mixed-type (MT) ones, which are comprised histologically of non-homogenous mixtures of IT and DT carcinomas. Stelzner and Emmrich [26] found that 28 MT carcinomas showed a deep infiltration of the gastric wall, frequent regional lymph node metastasis, and high staging in comparison to the other two types. Machado et al. [21] reported that inactivating E-cadherin mutations were exclusively observed in the diffuse component of the tumors, thus suggesting that MT carcinomas displayed phenotypic divergence. A recent study showed that bone-marrow-derived cells (BMDCs) could progress through metaplasia and dysplasia to intraepithelial cancer under the induction of the carcinogen, Helicobacter pylori [9]. Therefore, the analysis of distinct MT carcinoma components can be of remarkable pathogenic significance if it would be true that carcinoma cells originate from BMDCs. In the present study, 814 cases of gastric carcinomas were collected to further clarify the pathological characteristics of MT carcinoma and the gastric carcinogenesis pathways using a combination of tissue microarray (TMA) and immunohistochemical techniques.
Materials and methods
Subjects
A total of 814 gastric carcinomas were collected from surgical resection in Kouseiren Takaoka Hospital between 1998 and 2006. The patients with carcinomas included 573 men and 241 women (29–91 years, mean = 65.7 years). Among them, 312 cases were demonstrated with lymph node metastasis and 24 with liver metastasis. None of these cases underwent either chemotherapy or radiotherapy before surgery. All patients gave their informed consent for the use of tumor tissue specimens for clinical research and the University Ethical Committee approved the research protocol. All patients were followed up by consulting their case documents and through telephone interviews.
Pathology
All tissue specimens were fixed in 4% neutralized formaldehyde, embedded in paraffin, and cut into 4-μm sections. These sections were stained by hematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the TNM system of the Internationale Contre le Cancer (UICC) indicating the extent of tumor spread [25]. The histomorphological architecture of the tumors was expressed according to Lauren's classification [7, 16, 30]. The growth patterns were divided into five groups based on a modification of Emmrich's method [26]. Briefly, group I showed a combination of two components with diffuse distribution. Group II displayed both components with the border clearly visible. Group III represented some signet cells in the intestinal and diffuse components. Group IV were mucinous carcinomas with signet cells. Group V exhibited separately intestinal and diffuse components in the gastric wall. In addition, the depth of invasion, lymphatic and venous invasion, and peritoneal dissemination were all determined.
Tissue microarray
IT and DT carcinomas, as well as both components of the mixed type, were identified in HE stained sections of the selected tumor cases and a 2-mm-diameter tissue core of each donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Tokyo, Japan). Four-micrometer-thick sections were cut from the recipient block and transferred to poly-lysine-coated glass slides. HE staining was performed on TMA for confirmation of the tumor tissue (Fig. 1).
Fig. 1HE staining of TMA of gastric carcinomas
Immunohistochemistry
Consecutive sections were deparaffinized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by irradiating in target retrieval solution (TRS, DAKO, Carpinteria, UAS) for 15 min in a microwave oven (Oriental rotor Lmt. Co. Tokyo, Japan). Five percent bovine serum albumin (BSA) was then applied for 15 min to prevent non-specific binding. The sections were incubated with primary antibodies for 15 min, then treated with the anti-mouse or anti-rabbit Envison-PO (DAKO, USA) antibodies for 15 min. All incubations were performed in the microwave oven for intermittent irradiation as described previously [15]. After each treatment, the slides were washed with TBST (10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20) three times for 1 min. The primary antibodies are summarized in Table 1. All immunostaining was visualized with 3, 3′-diaminobenzidine (DAB) with 5-min reaction and counterstained with Mayer's hematoxylin. Omission of the primary antibody was used as a negative control.
Table 1 Primary antibodies used in this studyNamesSourceCompanyDilutionKi-67RabbitDAKO, Carpinteria, USA1:25Caspase-3RabbitDAKO, USA1:150p53MouseDAKO, USA1:100FHITRabbitNeomarkers, Fremont, USA1:200MaspinMouseNovocastra, Newcastle upon Tyne, UKRead-to-useEMMPRINMouseNovocastra, UK1:100VEGFRabbitLabvision, Fremont, USA1:50P-GSK3β-ser9RabbitSAT, USA1:300MUC-2MouseNovocastra, UK1:100MUC-4MouseNovocastra, UK1:100MUC-5ACMouseNovocastra, UK1:100MUC-6MouseNovocastra, UK1:100CD44MouseDAKO, USA1:50E-cadherinMouseTakara, Otsu, Japan1:100β-CateninMouseCalbiochem, CA, USA1:200
The immunoreactivity to Ki-67 and p53 was localized in the nucleus; FHIT, caspase-3, vascular growth factor (VEGF), maspin, phosphorylated glycogen synthase kinase 3β-ser9 (P-GSK3β-ser9), MUC-2, MUC-5AC, and MUC-6 in the cytoplasm; EMMPRIN and MUC-4 in the cytoplasm and membrane; CD44 and E-cadherin in the membrane; and β-catenin in the nucleus, cytoplasm, and membrane (Fig. 2). All evaluations were performed blindly by two independent observers (Takano Y and Zheng HC).
Fig. 2Immunostaining of TMA of gastric carcinomas. The immunoreactivity to Ki-67 (a) and p53 (c) was localized in the nucleus; FHIT (d), caspase-3 (b), VEGF (g), maspin (e), P-GSK3β-ser9 (h), MUC-2 (i), MUC-5AC (k), MUC-6 (l) were seen in the cytoplasm, EMMPRIN (f) and MUC-4 (j) were seen in the cytoplasm and the plasma membrane, CD44 (m), and E-cadherin (n) in the plasma membrane; and β-catenin (o) in the nucleus, cytoplasm, and plasma membrane. These molecules were strongly expressed in the intestinal-type gastric carcinomas
Statistical analysis
The statistical evaluation was performed using Fisher's exact possibility to differentiate the rates and the Mann–Whitney U test to differentiate the means of the different groups. Kaplan–Meier survival plots were generated and comparisons between the survival curves were made with the log-rank statistic. The Cox proportional hazards model was employed for multivariate analysis. p < 0.05 was considered to represent a statistically significant difference. The SPSS 10.0 software program was employed to analyze all data.
Results
Clinicopathological characteristics of gastric IT, DT, and MT carcinomas
As summarized in Table 2, 415 IT cases were identified (51.0%), 221 DT (27.1%), and 178 MT (21.9%) among the 814 gastric carcinomas in this study. Patients with IT carcinoma were found to be significantly older than those with DT (p < 0.05). The male and female ratios in the carcinoma cases gradually decreased from IT to DT through the MT groups (p < 0.05). It was the same for lower TNM staging in the three groups (p < 0.05). The positive rate of peritoneal spread was higher in the DT and MT cases than IT (p < 0.05). MT carcinomas showed aggressive characteristics, such as large size, deep invasion, frequent local invasion, and lymph node metastasis, compared to the IT and DT (p < 0.05). There was no significance between the three types of gastric carcinomas in the incidence of the liver metastasis (p > 0.05).
Table 2 Comparison of the clinicopathological features in gastric IT, DT, and MT carcinomasClinicopathological featuresIntestinal-type carcinomaDiffuse-type carcinomaMixed-type carcinomaCase number415 (51.0%)221 (27.1%)178 (21.9%)Age (mean ± SD, years)67.09 ± 10.7362.03 ± 11.3*65.80 ± 13.28Sex (male:female)324:91125:96124:54**Tumor size (mean ± SD, cm)3.82 ± 3.215.50 ± 6.955.56 ± 3.08**Invasion into muscularis propria153 (36.9%)121 (54.8%)123 (69.1%)**Lymphatic invasion (+)117 (28.2%)83 (37.6%)99 (55.6%)**Venous invasion (+)49 (11.8%)32 (14.5%)39 (21.9%)**Lymph node metastasis (+)113 (27.2%)94 (42.5%)105 (59.0%)**Liver metastasis (+)10 (2.4%)8 (3.6%)6 (3.4%)Peritoneal spread (+)14 (3.4%)***22 (10.0%)21 (11.8%)TNM staging (O, I)302 (72.8%)69 (31.2%)112 (63.0%)***p < 0.05 compared with intestinal- and mixed-type carcinomas**p < 0.05 compared with intestinal- and diffuse-type carcinomas with both analyzed***p < 0.05 compared with diffuse- and mixed-type carcinomas
Pathological behaviors of gastric MT carcinomas with different growth patterns or histological distribution of components
According to the growth pattern, the intestinal and diffuse components diffusely distributed without absolute border in group I of the MT carcinomas, which occurred more frequently (60.0%, 106/178) than the other four groups. As indicated in Table 3, most groups III and V gastric carcinomas were generally of early stage with less local invasion, infrequent lymph node metastasis and low TNM staging. In this study, only four advanced gastric carcinomas belonged to group IV, among which three cases were women and all were accompanied with lymphangiogenic invasion, lymph node metastasis, and high TNM staging. Although the main histological component was different from that in lymph node metastasis, the statistical data revealed that there was histological consistency between primary and lymph node metastasis foci (p < 0.05). There were no remarkable differences in the pathological behaviors of MT carcinomas with different ratios of intestinal and diffuse portions (p > 0.05).
Table 3 Pathological behaviors of gastric MT carcinomasClinicopathological featuresnSex (male)Invasion into MPLymphatic invasionVenous invasionLymph node metastasisStagingnI>DI=DI<D(O, I)Growth pattern I10673 (68.9%)82 (77.4%)64 (60.4%)26 (24.5%)69 (65.1%)32142335 (33.0%) II2518 (78.3%)21 (84.0%)16 (64.0%)7 (28.0%)16 (64.0%)5567 (28.0%) III2922 (75.9%)11 (37.9%)9 (31.0%)3 (10.3%)10 (34.5%)43318 (62.1%) IV41 (25.0%)4 (100.0%)4 (100.0%)1 (25.0%)4 (100.0%)0220 (0.0%) V1410 (71.4%)5 (35.7%)6 (42.9%)2 (14.3%)6 (42.9%)2049 (64.3%)Histological appearance Intestinal>diffuse8059 (73.8%)54 (67.5%)43 (53.8%)20 (25.0%)48 (60.0%)3081033 (41.3%) Intestinal=diffuse2015 (75.0%)12 (60.0%)9 (45.0%)5 (25.0%)10 (50.0%)3528 (40.0%) Intestinal<diffuse7850 (64.1%)57 (73.1%)47 (60.3%)14 (18.0%)47 (60.3%)10112628 (35.9%)MP Muscularis propria; in lymph node metastatic foci: I>D intestinal>diffuse, I<D intestinal<diffuse, I=D intestinal=diffuse
Immunohistochemical analysis of intestinal or diffuse components from three types of gastric carcinomas
Table 4 demonstrates that all the markers except MUC-5AC were expressed at higher levels in IT carcinomas than in MT ones (p < 0.05). The expression of maspin, EMMPRIN, VEGF, MUC-4, and E-cadherin was stronger in the intestinal component of MT carcinomas than in their diffuse counterpart (p < 0.05). Immunoreactivities to Ki-67, EMMPRIN, and VEGF were weaker in the intestinal-type carcinomas than in the intestinal component of the MT (p < 0.05), while it was the opposite for CD44, MUC-2, and MUC-6 (p < 0.05). In contrast, the diffuse components of the MT carcinomas showed greater expression of Ki-67, FHIT, VEGF, and P-GSK3β-ser9 than DT carcinomas (p < 0.05).
Table 4 Immunohistochemical analysis in gastric IT, DT, and MT carcinomasBiological markersIntestinal-type carcinomaMixed-type carcinomaDiffuse-type carcinomaIntestinal partDiffuse partKi-6788/146 (60.3%)*,**81/110 (73.6%)78/110 (70.9%)*57/119 (47.9%)Caspase-384/152 (55.3%)*52/119 (43.7%)37/119 (31.1%)24/115 (20.9%)p5376/151 (50.3%)*37/118 (31.4%)30/118 (25.4%)25/120 (20.8%)FHIT90/150 (60.0%)*63/113 (55.8%)48/113 (42.5%)*27/116 (23.3%)Maspin71/150 (47.3%)*55/112 (49.1%)***34/112 (30.4%)38/120 (31.7%)EMMPRIN87/147 (59.2%)*,**77/112 (68.8%)***43/112 (38.4%)32/117 (26.5%)VEGF95/151 (62.9%)*,**90/114 (78.9%)***61/114 (53.5%)*31/121 (25.6%)P-GSK3β-ser988/144 (61.1%)*74/113 (65.5%)67/113 (59.3%)*48/116 (41.4%)MUC-255/145 (37.9%)*,**31/118 (26.3%)27/118 (22.9%)23/117 (19.7%)MUC-458/153 (37.9%)*39/118 (33.1%)***21/118 (17.8%)18/118 (15.3%)MUC-5AC84/148 (56.8%)57/106 (53.8%)50/106 (47.2%)54/113 (47.8%)MUC-660/146 (41.1%)*,**26/117 (22.2%)18/117 (15.4%)9/123 (7.3%)CD4464/152 (42.1%)*,**32/114 (28.1%)31/114 (27.2%)32/120 (26.7%)E-cadherin91/142 (64.1%)*58/112 (51.8%)***38/112 (33.9%)43/119 (36.1%)Membrane β-catenin57/144 (39.6%)*36/117 (30.8%)26/117 (22.2%)26/118 (22.0%)*p < 0.05 compared with the diffuse-type carcinomas**p < 0.05 compared with intestinal-part carcinomas***p < 0.05 compared with diffuse-part carcinomas
Patients' outcome with different gastric carcinomas
Follow-up information for 500 carcinoma patients was used before 2002 for a period ranging from 5 days to 9.15 years (mean = 49.4 months). Figure 3 shows the survival curves stratified according to Lauren's classification. Kaplan–Meier analysis indicated that the patients with IT carcinoma had a higher cumulative survival rate than those with the DT and MT lesions (p < 0.05). Although the MT carcinoma patients' survival rate was comparatively lower than the DT ones, there was no statistical significance (p > 0.05). Multivariate analysis demonstrated that invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent factors for the poor prognosis of gastric carcinoma patients (p < 0.05; Table 5).
Fig. 3Relationship between prognosis of carcinoma patients' and Lauren's classification Kaplan–Meier curves of cumulative survival rate for the patients with gastric carcinoma according to Lauren's classification. IT Intestinal type, DT diffuse type, MT mixed typeTable 5 Multivariate analysis of the clinicopathological variables of gastric carcinomasClinicopathological parametersRelative risk (95% CI)p ValueAge (≥65 years)1.224 (0.849–1.764)>0.05Sex (male:female)1.255 (0.827–1.904)>0.05Tumor size (≥4 cm)1.466 (0.862–2.493)>0.05Invasive depth (Tis,1/T2,3)2.945 (1.533–5.657)<0.05Lymphatic invasion (−/+)1.394 (0.865–2.247)>0.05Venous invasion (−/+)1.692 (1.097–2.608)<0.05Lymph node metastasis (−/+)1.983 (1.111–3.537)<0.05Peritoneal spread (−/+)3.197 (2.031–5.031)<0.05Liver metastasis (−/+)5.248 (2.575–10.697)<0.05Lauren's classification (intestinal/diffuse/mixed)1.351 (1.084–1.683)<0.05CI Confidence interval
Discussion
Histologically, Lauren's classification of gastric carcinomas is based on the morphological appearances. IT carcinomas are characterized by cohesive carcinoma cells forming gland-like tubular structures with an expanding or infiltrative growth pattern, like a papillary and well-differentiated adenocarcinoma. However, the cell cohesion is less apparent or absent in DT carcinomas, and cancer cells diffusely spread in the gastric wall as poorly differentiated adenocarcinomas, SRC, or undifferentiated carcinomas [23, 30]. However, there are two types of histological distribution in the MT carcinoma, as described by Lauren [16]. In this study, carcinoma diagnosed as MT made up 21.9% of the whole series in this population, more than that reported by Borch et al. (13%), Lauren (14%), and Stelzner and Emmrich (14.1%), and less than that reported by Carneiro et al. (38.5%) [2, 5, 16, 26]. This discrepancy might be due to the differences in diagnosis criteria, geographical regions, human races, living habits, and so forth. The age and sex distribution of the patients with MT carcinoma was intermediate although IT carcinomas frequently occurred in old men, compared with DT carcinomas.
Morphologically, there are both intestinal and diffuse components in MT carcinomas, and they were found to be significantly larger, more advanced, and more frequently spread into local lymphatic and venous vessels or regional lymph nodes, compared with IT and DT carcinomas in the present study, consistent with other reports [6, 16, 30]. Kozuki et al. [14] found that prominent lymphatic permeation and lymph node metastasis were more frequently observed in MT than in the pure type of gastric carcinomas. It was previously documented that the MT carcinomas showed a deeper infiltration of the gastric wall, a higher metastatic rate to the regional lymph nodes, and the need for higher staging with the TNM system of UICC classification, in contrast to IT and MT carcinomas [26]. These findings suggest that the pathobiological characteristics of the MT carcinomas were more aggressive than the other two types, which accounted for their comparatively poor prognosis observed in the present study although there was no statistical significance. Among the MT carcinomas, five groups were classified according to their growth pattern. Half of the present cases belonged to group I, which means there was diffuse distribution of the intestinal and diffuse component without clear borders in most of the MT cases. In general, groups III and IV of MT carcinomas did not exhibit deep invasion, comparatively high staging or frequent spreading, compared with other types. However, group IV of carcinomas with mucin and signet cells was more advanced with wide spreading, including local vessels and lymph node metastasis. Therefore, surgeons should carefully treat the patients with MT carcinomas in clinical practice because of their aggressive characteristics and poor prognosis, especially those classified as group IV. Furthermore, it was found that the major component in the MT primary foci morphologically paralleled to that in the corresponding lymph node metastasis although the converse situation also appeared. This inconsistent phenomenon could be explained by sampling bias or the metastatic potential of individual carcinoma cells.
Compared with IT carcinomas, the MT counterpart always exhibited more severe characteristics, including invasion into muscularis propria, lymphatic invasion and lymph node metastasis, and high TNM staging in the present study, as observed by Lauren and other studies [16, 26, 30]. To clarify the pheno/genotypes of gastric carcinoma, the expression of Ki-67, caspase-3, p53, FHIT, maspin, VEGF, EMMPRIN, P-GSK3β-ser9, MUC-2, MUC-4, MUC-5AC, MUC-6, E-cadherin, and β-catenin was examined by immunostaining. p53, FHIT, and maspin as tumor suppressor genes play important roles in regulating the balance between the proliferation and apoptosis of cancer cells [25, 30, 31, 34, 35]. Ki-67 antigen is present in the nuclei of cells undergoing proliferation and should be regarded as a good marker for cell proliferation [34]. Caspase-3 is responsible for the cleavage of poly (ADP-ribose) polymerase, actin, and sterol regulatory element binding protein and reflects the apoptotic level as a key protease in the cascade reaction of the apoptotic pathway [28]. In vivo and vitro evidences indicated that EMMPRIN and VEGF are involved in angiogenic processes in malignancies [29, 32]. Glycogen synthase kinase-3β (GSK3β) belongs to the serine/threonine protein kinase family and is also involved in regulating the balance between proliferation and apoptosis and can be inactivated via ser-9 phosphorylation by p70 S6 kinase, p90Rsk, Akt, certain isoforms of proteins kinase, and cyclic AMP-dependent protein kinase [1]. MUC-2, MUC-5AC, and MUC-6 are markers for intestinal goblet cells, superficial epithelium, and gastric pyloric gland cells, respectively, which can reflect the mucin secretion and variations in gastrointestinal malignancies. MUC-4 is a heterodimeric glycoprotein complex and expressed in several human epithelial carcinomas [3, 33]. CD44 is a cell surface glycoprotein involved in cell/cell and cell/matrix interactions. CD44 overexpression has been linked to the growth and spread of a range of different types of malignancies [8]. The E-cadherin can interact with β-catenin to form a complex, which is closely linked to cell adhesion and differentiation [19, 27, 36]. In the present study, all of the markers except MUC-5AC showed greater expression in the IT than in the DT carcinomas. Although some evidences indicate that MUC-5AC was strongly expressed in gastric DT carcinoma, in comparison with IT ones, there was no statistical difference in line with our finding [20, 22]. These data suggested that there were significant differences in the proliferation, apoptosis, angiogenesis, mucin production, and cell adhesion between the IT and DT carcinomas and these molecules mechanistically contributed to the molecular distinction in the morphological, behavioral, and histogenic aspects between both types of gastric carcinomas.
In general, DT carcinoma is believed to derive de novo from the peripheral stem cells of gastric gland neck proliferative zone without a recognizable precursor lesion except hereditary diffuse gastric cancer because in situ carcinoma or globoid dysplasia is its precancerous lesion, respectively, according to the Chinese and Western pathologists' opinions [4, 5, 9, 18,30]. When long-standing gastric inflammation causes tissue injury and stem cell failure with time, BMDCs are recruited and engrafted into the tissue stem cell niche, where BMDCs can behave in a way indistinguishable from endogenous tissue stem cells. With continued inflammation and injury, they can undergo sustained proliferation and malignant transformation into IT carcinomas, passing through precancerous stages of metaplasia and dysplasia when genetic defects, such as mutation, deletion, or rearrangement, are accumulated resulting in corruption of the balance between proliferation and apoptosis [17]. Likewise, dedifferentiation of intestinal to diffuse carcinoma had been identified as another histogenic pathway according to the histological heterogeneity of tumor cells, especially in the carcinomas containing moderately and poorly differentiated components with similar morphological appearance and diffuse distribution. If so, it is possible that the distinct components in MT carcinomas arise from the stem cells with common genetic traits and follow different carcinogenic pathways. Conversely, diffuse carcinoma could be derived from heterogeneity of the intestinal counterpart. According to our present data, no differences in the expression of Ki-67, caspase-3, FHIT, CD44, P-GSK3β-ser9, MUC-2, MUC-5AC, MUC-6, and membrane β-catenin were observed between the intestinal and diffuse components of MT carcinomas, which supports the possibility of similar origin or dedifferentiation. However, increased expression of EMMPRIN, VEGF, MUC-4, and E-cadherin in the intestinal component compared to the diffuse counterpart also provided evidence that the original carcinoma cells might undergo distinct carcinogenic routes resulting in the morphological distinction of both components. Both the intestinal and diffuse components in MT carcinomas had increased expression of Ki-67, EMMPRIN, and VEGF and reduced E-cadherin. Serum VEGF levels were found to be significantly higher in patients with MT gastric carcinomas than those with pure lesions [10]. It was proposed that MT carcinomas biologically displayed more aggressive behaviors than other types, including decreased cell adhesion, increased proliferation, and angiogenesis.
Most of the evidences in the present data indicated that the pathological behaviors of MT carcinomas were more aggressive than the other types, which was closely linked to the prognosis. It was previously documented that the MT patients' survival is significantly worse than those with IT or DT carcinomas regardless of their location [5]. However, this study demonstrated that there was no significance between the MT and DT carcinoma patients' survival rates although the former was lower than the latter. Additionally, both survival rates were lower than that with IT carcinomas. To avoid sampling bias from a shorter follow-up time, the older cases were chosen for the survival analysis, but they yielded consistent results. Furthermore, Cox's hazard proportional analysis indicated that the invasive depth, venous invasion, lymph node, peritoneal or liver metastasis, and Lauren's classification were independent prognostic factors for gastric carcinomas.
In summary, IT carcinoma, which is positively correlated with favorable prognosis, frequently displayed high levels of proliferation, apoptosis, angiogenesis, mucin production, and cell adhesion. Gastric MT carcinoma showed more aggressive behaviors than IT and DT ones. There was a significant difference in the proliferation, apoptosis, angiogenesis, mucin secretion, and cell adhesion between the IT and DT carcinomas, whereas only a few characteristics were differentially detected in the intestinal and diffuse component of the mixed-type carcinoma, suggesting that different components of MT carcinoma might originate from common stem cells, but follow distinct histogenic pathways. Furthermore, these results confirm that Lauren's classification is significant regarding the histopathogenesis and differentiation and considered as a guide to the clinical treatment of gastric carcinoma.

gastric carcinoma
pathobiological behaviors
carcinogenesis
lauren's classification
mixed type