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Laminin.png


Introduction

Laminin (LM) is a glycoprotein, found within the extracellular matrix [1], of many embryonic and adult tissues [2]. LMs have a cross – shaped structure, formed by three short arms (alpha, beta, and gamma chains) and one long arm which is a globular domain that binds to cellular receptors[3]. LMs are the building blocks for cellular networks bridging the intracellular and extracellular compartments of the basement membranes (BM) [4]. They regulate the development and function of numerous tissues and organs [5], playing an important role in tissue homeostasis and maintenance[6]. LMs are also believed to play a role in cancer progression [6], by contributing to tumour dissemination and metastasis in advanced breast carcinomas as well as other tumor types [7]. Furthermore, LMs are believed to influence adipose tissue structure [3], have a role in the blood – brain barrier [8] and also regulate cellular processes, such as cell migration, differentiation and proliferation[9].


Defects in laminin chains can result in:

  • Ocular diseases, such as in the development of the eye [10]
  • Epidermolysis Bullosa, a disorder resulting in structural weakening of the skin and mucous layer [11]
  • Muscular dystrophy, which is a group of disorders characterised by the weakening of the skeletal muscle [12]


Current and potential investigations into laminin include:

  • The regenerative ability of human adipose tissue derived from stromal cells, which is currently being investigated in the field of regenerative medicine [13]
  • The effects on individual truncated LM chains in the absence of their normal partner [9]
  • And possible therapeutic solutions for advanced cancer patients [7].


<mediaplayer width='500' height='300'>https://www.youtube.com/watch?v=QGnFgDEWCLs</mediaplayer>[14]

Video 1. An introduction to laminins.

This page will discuss the structure of LM, the general functions of LM as a whole in the ECM, and some of the functions of most of the LM heterotrimers. Due to the lack of research on some of the isoforms, there is insufficient information to determine their function. We discuss the functions of five laminin isoforms; laminin-111, laminin-211, laminin-332, laminin-411, and laminin-511 and also uncover the effect of LM abnormalities, specifically in cancer, epidermolysis bullosa, muscular dystrophy, and development. The current research on LMs ranges over a broad spectrum. The main area of concern is LMs' role and contribution to cancer. However, LMs role in other research areas, such as in skin blistering, anti – adhesive properties and presence at the dento – gingival interface, has also been investigated.

History

Graph 1 The number of publications on Laminin, per year, from 1975 to 2014. A rapid increase in publications can be seen from 1979 onwards, when Laminin was first discovered.

First Laminin Isoform Discovered

In 1979, a large, non - collagenous glycoprotein was isolated from both an Engelbreth-Holm-Swarm (EHS) sarcoma and basement membrane producing cells[4].

Experimental Process

In a transplantable mouse tumor, the EHS sarcoma, which produces an ECM of BM, was found to be a convenient source for the isolation of the collagenous component, excluding the capsule, because tumor proteins are made up of approximately 5-10% of type IV collagen.

Using neutral buffers little amounts of this collagen can be extracted. However, with dilute acetic acids, collagen can be extracted from the tumours found in lathyritic mice. Neutral buffers though, are still able to extract large amounts of non - collagenous proteins.

Following, it was purified in quantities sufficient for biochemical, structural and immunological characterisation, and given the name laminin.

Laminin was concluded as a major component of the tumour matrix and was localised to the basement membrane in normal tissues by immunological techniques[15].

Of the extracts, a large component was found to be a high molecular weight non-collagenous glycoprotein, which they named ‘laminin’[15].

Following, research focused on laminins has greatly increased, as seen in Graph 1.


Findings

Biochemical analysis revealed that the LM structure consisted of three linked polypeptides, which were given the names A, B1 and B2.

Electron microscopy observations revealed that these three chains formed an asymmetrical cross-shaped structure. This cross-shaped structure consisted of a long arm and three short arms.

As a result of further discoveries of various cells and tissues, cloning and automated sequencing techniques, genetically different LM subunits in humans and other species were identified.

This highlighted that the LM molecule from the EHS sarcoma was not unique, but part of a new protein family. Hence, in order to distinguish between the diverse members, the LMs were named and numbered in order of their discovery[4].


Unifying Nomenclature Introduced

In 1994 (as reviewed in [16]), the consecutive A, B1 and B2 chains were re – named with the Greek letters, α, β, and γ, respectively.

Because it was noticed that the sequencing of the subunits provided evidence for distinct polypeptide sequences at the amino acid level, they were named by the addition of numbers to the Greek letters[4].


Laminins Today

Today, LM isoforms are known by their chain compositions. The α1, β1, and γ1 chain is known as laminin 111. This chain is the prototype of the laminin family and is the best characterized laminin isoform[4].

Structure

Figure 1 The laminin structure is composed of three polypeptides chains named A, B1 and B2 (nowadays α, β and γ chains, respectively) which are held together in cruciform-like structure by disulphide bonds (three disulfide-linked polypeptides).Red is collagen binding, blue is entactin binding and black is proteoglycan binding.

Laminin is a mosaic glycoprotein of the ECM of metazoans composed of different domains with different structures and functions [17]. According to H.K. Kleinman et al. laminin has multiple structures in different tissues due to variations in the number and type of chains. The LM structure is composed of three polypeptides chains named A, B1 and B2 (nowadays α, β and γ chains, respectively) which are held together in cruciform-like structure by disulphide bonds (three disulfide-linked polypeptides). H.K. Kleinman et al. explains the A chain has three globular domains at the amino terminus separated by three epidermal growth factor (EGF)-like repeats. The B1 (or β) and B2 (or γ) chains are structurally similar but contain only two amino terminal globules and EGF-like repeats and lack the globule at the carboxyl end [18]. Furthermore, observations indicated that the chains form an asymmetrical cross-shaped structure, with a long arm of about 77 nm carrying a large globule at its end, and three short arms, two of 34 nm and one of 48 nm, each being terminated by a globular domain [19].The human genome encodes 11 genetically distinct LM chains differing at the level of the amino acid sequences. However, the polypeptide chains in a cross-shaped pattern is conserved among laminin isoforms. [4] Figure 1 is an example of a basic laminin structure.


Structure and ECM Interactions

The laminin structure has an important role in the extracellular matrix interactions. The heterotrimeric structure is one of the major components of basement membrane (BM) and it is critical to assemble the cell network. The three arms of the laminin form the network nodes and all the arms are required (α, β and γ arm) for it.[3]

-The globular domain of the long arm binds to cellular receptors, such as integrins, heparan sulfates, α-dystroglycan, sulfated glycolipids.[3]

-The proteoglycans (perlecan and agrin) provide the collateral anchorage of the laminin network.[3]

-The type IV collagen interacts with the laminin network through the heparan sulfate chains (perlecan and agrin) and entactin (nidogen), forming a second network (See Figure 1).[3]

Furthermore, it is interesting to mention that the difficulty of imaging BM in situ prevents a direct understanding of BM architecture, because the process of imaging involves a harmful extraction from tissues. Therefore, the current model is more based on indirect observation that involves multitude clues than direct observation. From these observations, some questions are raised: the dimension of thickness of a BM is of the same order as a single laminin molecule, which makes unlikely that laminins are erected on the surface of cells. Finally, the researchers are not sure if they know all of the important interactions between laminin and the BM assembly and maturation. One reason for this is because some of current understanding about the interactions is based on early experiments with crude protein preparations. Then, the modern recombinant and proteomic techniques might be useful for the future researches in additional laminin interactions.[3].Figure 2 is an example of laminin interactions.

Figure 2 Schematic drawing of the laminin-111 heterotrimer. The three short arms of the cross-shaped molecule have a common domain structure and consist of laminin N-terminal (LN) domains, laminin-type epidermal growth factor-like (LE) domains, and L4 domains, as indicated for the α1 chain. The α1 chain uniquely contains five laminin G-like (LG) domains. LG1-3 likely interact with the C-terminal residues of the γ1 chain. (B) The three-arm interaction model of laminin self-assembly. The ternary nodes in the network are formed by the N-terminal regions of one α, one β and one γ chain. The long arm of the laminin heterotrimer is not involved in network formation[3].


The recent researches found relations between LM structure and its influence in phenotypes. The research "Laminin α4 deficient mice exhibit decreased capacity for adipose tissue expansion and weight gain” shows an understanding of the mechanisms related to extracellular matrix (ECM) and regulation of adipose tissue expansion. The research describes the role of laminin α4 (ECM protein that surrounds adipocytes) on weight gain and adipose tissue function. Two mice were examined in adipose tissue accumulation, lipogenesis, and structure. The mice with a null mutation of the laminin α4 gene (Lama4-/-) exhibited reduced weight gain in response to both age and high fat diet when compared wild-type (Lama4+/+) control animals. These results suggest that laminin α4 influences adipose tissue structure and function. Furthermore, it could be applied in therapies that reduce obesity morbidity and mortality[5].

Function

Laminin is the major component of the basal lamina, the extracellular matrix (ECM) which surrounds all epithelia, muscle, peripheral nerve and fat cells. Then it is related with cell adhesion, migration, growth, differentiation, neurite outgrowth and tumor metastases and its structural level is now well characterized[20]. Laminins play a role in BM assembly, adhesion [21], cell proliferation, migration, and differentiation[22][1]. They control cellular behaviour by interacting with cell surface receptors (e.g. integrins, syndecans, and α-dystroglycans) [23]. The interaction between integrins and LMs causes cells to be anchored to the BM and initiate intracellular signals [24]. As reviewed in [4], LM has 2 terminal ends, the C-terminal end and the N-terminal end. The N-terminal end is responsible for interacting with the different ECM proteins and is therefore important for the assembly of the BM. The C-terminal end is involved with the interaction with cell surface receptors. Therefore, this terminal end is responsible for cellular adhesion, migration, signalling, and survival.

The different combinations of the three chains of LM form different LM heterotrimers, or better known as isoforms. Each LM isoform has different functions in the body. Furthermore, the function of each isoform can vary depending on the environment they are in, i.e. the type of cell or tissue where they are located [23]. For example, laminin-111 in muscle promote the regeneration of muscle cells [22] whereas in human mesenchymal stem cells, they promote the outgrowth of neurites [25].

Table 1. The functions of some of the different laminin heterotrimers.

Laminin Heterotrimer
Function
Laminin-111
involved in regulating muscular morphogenesis by promoting regeneration in muscle cells[22][26]; promotes neurite outgrowth in human mesenchymal cells via activation of FAK and MAPK cascades[25]
Laminin-211
essential for the contractility of airway smooth muscle cells[27][28]
Laminin-121
has a strong affinity for α7β1 integrin, which promotes neurite outgrowth [29]
Laminin-221
involved in the promotion of neurite outgrowth in motoneurons, particularly its laminin β2 subunit[30]
Laminin-332
involved in the synthesis of tyrosine, which increase the level of melanin produced by melanocytes[31]
Laminin-311
involved in the maintenance of the alveolar tissue architecture [32]
Laminin-321
works in conjunction with laminin-332 in mediating epithelial cell adhesion to the BM[33]
Laminin-411
has a vascular role; increases the migration and adhesion of human dermal microvascular endothelial cells (HDMECs), which promote the tubule formation of endothelial cells[34]
Laminin-421
binds with α6β1 integrins to control the migration of mesangial cells induced by insulin-like growth factor binding protein-5 (IGFBP-5)[35]
Laminin-511
a potent substrate for up regulating α3β1 integrin, which is a promoter for hair growth[36][37]
Laminin-521
essential for the formation of the glomerular filtration barrier (as reviewed in [38]; also involved in preventing Schwann cells from invading the synaptic cleft in a neuromuscular junction [39]
Some laminins in detail

Laminin-111

Laminin 111 (LM-111) is the one of the first, and predominant, ECM proteins expressed during embryonic development[22][21]. LM-111 provides structural support to the BM[21]. More importantly, this LM isoform’s main role is to regulate muscular regeneration[22][26]. LM-111 is known to prevent the effects of muscular dystrophy, such as skeletal muscle weakness due to damages in cells and tissues[22][26]. It does this by playing a role in myoblast fusion[22], the formation of new and larger myofibres, and acting as a substitute to LM-α2 deficiency[26]. The injection of LM-111 helps re-establish the sarcolemma in muscle cells that are laminin-a2 deficient[26]. This LM isoform also increases muscle integrity by interacting with a7B1 integrin and a-dystroglycan[26], which improves the number of satellite cells by improving satellite cell proliferation[22][26]. LM-111 provides a link between the ECM and the sarcolemma, which prevents muscle degeneration and apoptosis, and promotes muscular adhesion to the basal lamina[26]. There is evidence showing that LM-111 prevents muscle damage. In postexercise mice, those that were treated with the laminin isoform had fewer muscle fibres with centrally located nuclei, which is commonly a sign for muscle damage[22]. LM-111 also plays a role in the epithelial-to-mesenchymal transition (EMT) in cells. A fragment of the LM molecule interacts with matrix metalloproteinases (MMPs), specifically MMP2. The LM-111 fragment down-regulates MMP2, which in turn lowers the amount of BM undergoing degradation[21].

Laminin-211

Laminin 211 (LM-211) plays a role in the contractility of airway smooth muscle (ASM) cells, which relates to the pathology of asthmatic attacks. The expression of this LM isoform in ASM cells is essential for its contractility as well as reducing the levels of proapoptotic proteins in ASM cells [27][28], which results in the hypertrophy of these cells[27]. This relates to asthma because asthmatic airways are characterised to have ASM cells with increased contractility[28]. LM-211 also has a role in the control in the interactions between thymocytes and thymic epithelial cells (TECs). The deposit of LM-211 creates a microenvironment for thymocytes to proliferate, maturate, differentiate, and migrate. Furthermore, the thymocyte-TEC interaction was blocked when antibodies specific for LM-211 were introduced[40].

Laminin-332

Melanocytes are cells that synthesise melanin, a component of the pigmentary system of the skin. The melanin, produced by melanocytes, migrate to keratinocytes for protection. Laminin 332 (LM-332) is derived from keratinocytes and plays a role in the migration and adhesion of melanocytes[31][41]. LM-332 is also promotes the synthesis of melanin in melanocytes by controlling the levels of tyrosine[31]. Tyrosine is essential for the synthesis of melanin and it's level is proportional to the level of melanin i.e. an increase in the level of tyrosine will also increase the level of melanin made[41]. This links to LM-332 because this LM isoform increases the intracellular tyrosine levels and also increases the uptake of extracellular tyrosine[41]. However, the mechanism behind the control of tyrosine levels by LM-322 is still unknown.

Laminin-411

Laminin 411 (LM-411) is expressed in the endothelial BM and plays a significant role in the maintenance of the vascular endothelial BM[34]. Although, Laminin-511 maybe expressed in vascular endothelial BMs as well, LM-411 is expressed in all BMs of all blood vessels, whereas Laminin-511 is only expressed in capillaries and some veins and venules[42]. This LM heterotrimer is involved in the survival of endothelial cells, particularly in their migration and adhesion[34]. The presence of LM-411 increases the migration and adhesion of human dermal microvascular endothelial cells (HDMECs). In turn, the migration of HDMECs promotes tubule formation of endothelial cells[34]. Therefore, it can be deduced that this laminin heterotrimer is involved in vascular formation. Therefore, a lack in LM-411 expression leads to disrupted vascular endothelial BMs. In a study, the lack of LM-411 expression resulted in a bleeding phenotype in newborn mice[42]. LM-411 is related to type IV collage, in that the overexpression of LM-411 increases the number of type IV collagen in capillary BM. The absence of LM-411 leads to no formation of type IV collagen, which affects the assembly of the BM[34].

Laminin-511

Laminin 511 (LM-511) is one of the first ECM proteins expressed during embryogenesis[22]. LM-511 is another LM heterotrimer, release by keratinocytes[43], associated with hair downgrowth[44][43]. Lm-511 is found in the BMZ and interfollicular epithelia but it is most numerous in the BMZ, specifically around the hair follicles[44]. Unlike LMm-332, LM-511 is the primary LM [37] for the stimulation of hair growth and elongation[36]. LM-511 is associated with α3 integrin, in terms of regulating hair growth[44]. α3 integrin acts as a promoter of hair growth[44]. This links back to LM-511, which is a potent substrate for the upregulation the expression of the α3β1 integrin[36][37][43]. Therefore, the presence of LM-511 promotes hair growth via α3β1 integrins[36][37]. Another process as to how LM-511 affects hair follicle involves dermal blood vessels. α3 integrin is also involved in the migration of endothelial cells. Therefore, it is plausible that LM-511 is associated with the recruitment of dermal blood vessels essential for the formation of hair follicles[37]. LM-511 is also involved in the formation of the BMZ. The lack of LM-511 results in a discontinuous lamina densa, which hinders the downgrowth of the hair follicles[43].

Together with LM-411, LM-511 also plays a role in the maintenance of the vascular endothelial BM[42]. Unlike LM-411, LM-511 is expressed only in capillaries and some venules and veins. However, the failure to express LM-411 leads to compensation by LM-511. Due to this event, angiogenesis is increased, which conseuquently enhances the growth of tumours and finally metastasis. Therefore, this LM heterotrimer, even if not directly, is also involed in angiogenesis, tumour formation and metastasis[42].

Abnormalities

Cancer

Some Laminin isoforms have long been implicated in tumor invasiveness and metastasis. It was found that the majority of these laminin chains are the same isoforms in normal epithelial. [45]. Past research has manly been completed on laminin 332 and laminin B1 (LAMB1) however other laminin subunits have been implicated. These include Laminin 511and Laminin 5 alpha 2.


Laminin 332:
Figure 3: Internal cascade from laminin 332 to migration of cell

Laminin 332 (LN332) is a basally expressed extracellular matrix protein that demonstrates tumour-promoting properties. In normal tissue Laminin 332’s main role is to maintain epithelial-mesenchyme cohesion when the tissue is exposed to external disruptive forces. This is particularly evident in skin and stratified squamous mucosa[46]. It also stimulates cells including carcinoma cells to migrate allowing for metastasis and has thus been associated with the progression of a tumour[47]. This is depicted in figure 3, which illustrates the focal adhesion complex. Consisting of a range of proteins including α6β4 and α3β1 integrins, VII collagen and laminin 332, the complex involves an intracellular cascade. The association of laminin 332 with 31 integrin promotes RHOA GTPase activation leading to a non-directional mode of cell migration. The binding of laminin 332 to 64 integrins promotes RAC1 GTPase activation and a directional mode of cell migration. This cascade promotes tumour invasion and cell survival[48].


Laminin beta 1 (LAMB1):

Epithelial-to-mesenchymal transition (EMT) is a process allowing an epithelial cell to assume a mesenchymal phenotype through biochemical changes via its basement membrane. This process is essential for cell migration and gives carcinoma cells invasive properties. [21]. Laminin, beta 1 is expressed in most tissues that produce basement membranes[21]. Mapping of 5'-untranslated region (UTR) of Laminin-B1 indicates that it contains internal ribosome entry site (IRES) between −293 and −1, upstream of the start codon. It has been reported that the IRES mediates translation of LamB1 during EMT[49]. A recent study found that miR-124-5p, a sequence in the LAMB1 chain functions as a tumour suppressor and could function as a molecular marker for glioma diagnosis and as a potential therapeutic[50].


Other laminin isoforms and subunits implicated in cancer:

Laminin Role
Laminin γ2 The cleaved-off Laminin γ2 short arm stimulates migration as a soluble factor [51].Furthermore it was shown that gastric carcinoma cells in humans produce and release monomeric Laminin γ2.
Laminin alpha 4 The alpha 4 subunit is a Constituent of Laminin 411 and Laminin 421. Recent studies suggest that the alpha4 chain has de-adhesive functions. This means they could play a role in detachment, migration and invasion of renal carcinoma cells.[52]
Laminin 111 A recent study showed that high levels of laminin-111 could deplete nuclear actin and induce cell quiescence [53].
Laminin alpha 3 Past studies have highlighted the attachment of Laminin α3 to tumor cells. This attachment leads to the prevention of these tumor cells from metastasizing[54].
Laminin 511 One of the more recent laminin isoforms to be identified is Laminin 511. It has been identified as a potent adhesive and pro-migratory substrate in both normal and tumor cells. A recent study examined the effect of suppressing Laminin 511, indicating a loss of adhesion and directional migration as a result. This Is illustrated in video 5.1. In this video the loss of adhesion and directional migration as the percentage of laminin α5 mRNA and protein suppressed is increased. [7].
Laminin 5 gamma 2 Laminin-5γ2 chain expression has been associated with many squamous cell carcinoma. A 2012 study found an associated with Laminin-5γ2 and tumor cell invasiveness and prognosis of lung carcinomas [55]. While a more recent study found and associated between Laminin-5γ2 and the intensity of tumour budding and density of stromal myofibroblasts in oral carcinomas[56].


<mediaplayer width='500' height='300'|centre|thumb|>https://www.youtube.com/watch?v=5Vg-dzM5tnU</mediaplayer>Video 2: Suppression of Laminin 511 results in loss of directional migration: Part 1- Control cells (Sc.5), Part 2- Cells (KD4.2 exhibit 59% suppression of laminin α5 mRNA and 24% suppression of α5 protein, Part 3- Cells (KD2.4) exhibit 57% suppression of laminin α5 mRNA and 75% suppression of α5 protein.[57]



Epidermolysis Bullosa

Figure 4: Clinical manifestations of Junctional Epidermolysis bullosa (JEB)[58]

An interconnected network maintains the stable association between the epidermis and dermis giving the skin strength. This network comprised of hemidesmosomes, anchoring fibrils and filaments require functioning units to maintain this stability. When this is not achieved diseases comprised of fragile skins arise, such as Epidermolysis bullosa.


Epidermolysis bullosa is caused by a mutation in Laminin-332 commonly know as laminin-5. Mutations in the genes that code for the three chains in laminin-332, namely LAMA3, LAMB3, LAMC2, lead to a disruption in the anchoring between the lamina Lucida and lamina densa. While mutations can occur in any of the three polypeptide subunits, mutations in LAMB3 are the most common accounting for approximately 80% cases [59]. Normally in the cutaneous basement membrane zone (BMZ) the transmembrane protein α6β4 integrin located in hemidesmosomes enters the basal lamina via its extra cellular domain. This region of the α6β4 integrin interacts with type IV collagen suprastructure containing laminin-332. These Laminin- 332 molecules act as anchoring filaments holding the integrin molecules down toward the basement membrane.This can be seen in figure 5. A defect in the laminin-332 causes the detachment of these layers. As a result the skin becomes fragile and minor friction can cause a separation between the lamina Lucida and lamina densa layers. The result of this is incredibly painful blisters[60].


Figure 5: Cutaneous basement membrane zone (BMZ)



Characteristic feature of Junctional Epidermolysis bullosa:

  • Scarring alopecia
  • Malformed fingernails and toenails
  • Aplasia cutis congenital- a congenital localized absence of skin
  • Milia- raised bumps on the skin
  • Irregular tooth enamel




Figure 6: Histological representation of blistering[58]



Congenital muscular Dystrophy

Figure 7: Laminin alpha interactions-2 interacting with the glycans of alpha α-dystroglycan[58]

Congenital Muscular dystrophy is a group of heterogeneous disorders characterized by the weakening of the skeletal muscle. Over 30 mutated genes, many of which encode for molecules involved in maintaining structural integrity and cell adhesion, can cause these muscular disorders. One of the most severe forms of muscular dystrophy is merosin-deficient congenital muscular dystrophy type 1A (MDC1A) and involves the mutation of laminin α2 or LAMA2 [62]. MDC1A is most common in Europe accounting for 30–50% of all congenital muscular dystrophies. [63]. Mutations on LAMA2 have been reported to range from absence of laminin α2 to the partial deficiency and are localized on the chromosome 6q22–23[12].The α2 chain is a constituent of laminin 2 (merosin) and is expressed in the basal membrane covering muscle fibers and the endoneurial basal membrane covering Schwann cells[64].

Laminin 2 interacts with dystrolgycan, which is intracellularly associated, with dystrophin. In the normal dystrophin-glycoprotein complex (DGC) the laminin protein binds to α-dystroglycan via the glycan chain attached. This occurs at the C-terminal end of the α chain, which contains globular G domains composed of 5 modules[65]. Dystrophin connects the cytoskeletal to the extracellular matrix and regulates calcium levels [66]. When these interactions are comprised dystrophin is effected and muscular dystrophies arise.


Characteristics of congenital muscular dystrophy:


  • Weakening of the muscles
  • joint contractures
  • White matter abnormalities
  • Respiratory problems[67]



Development

Laminin/ Laminin chain
About:
Abnormalities:
Laminin- 111 Laminin-111, a trimeric basement membrane glycoprotein, is one of the first extracellular matrix (ECM) proteins expressed during the two-cell stage in early embryogenesis. It is together with other proteins, collagen IV, nidogen and perlecan, an important aspect in the assembly of the basement membrane. With the β1 and γ1 chains of Laminin 111 no basement membranes can form.[68]. A lack of either of these chains or Mutations in them can lead to early post implantation lethality[69].
Laminin α5 chain The LM α5 chain is the most widely distributed LM chain. It is incorporated into the BMs before gastrulation. [70].The effects of LM α5 can be seen in figure 8 Studies that have been performed on mice indicate a deficiency in the LM α5 chain leads to an arrest in development due to failure of multiple organs at mid gestation. These included exencephaly, syndactyly, and placentopathy[71].
Laminin α2 chain The LM α2 chain is expressed in developing muscles form E11, during embryogenesis[12]. A deficiency of LM α2 in mice leads to the development of muscular dystrophy leading to death around 5 weeks after birth.[12].
Laminin α4 chain The LM α4 chain is a constituent of laminin 8 and 9 and is present in late embryonic and neonatal vascular basement membranes. It plays a vital role in the growth of micro vessels[72] Studies indicate Hemorrhages in alpha4-null mice appeared during the embryonic and neonatal period. In this particular study approximately 20% of the mice died within the first 2 days after birth[72].


Figure 8: Laminin alpha 5- Role in development [73]

Current Research

The Heterotrimeric Laminin Coiled – Coil Domain Exerts Anti – Adhesive Effects and Induces a Pro – Invasive Phenotype [9]

Study: To determine what would happen to individual truncated LM chains in the absence of their normal partners.

Method: A transfectable cell line with low endogenous LM chain expression was selected, by first encoding the truncated LM α1, β1 and γ1 chains which encode the entire laminin coiled - coil domain (LCC). See Figure 9.

Figure 9 Structure of Laminin - 111 domain organisation and recombinant laminin coiled - coil domain.

Results: Truncated α1 chains were found to be efficiently secreted as a monomer when expressed alone, as opposed to the β1 and γ1, which were poorly secreted. But, when all three were expressed together, they formed a trimer that was secreted into the medium. This confirmed, consistent with previous findings, that a chain subunit expression is necessary for the secretion of the β1 and γ1 chain partners and that the truncated chains can also assemble into the trimetric coiled - coil structure independent of the rest of the molecule. It was also revealed and demonstrated that the LCC domain inhibits cell adhesion and spreading, however anti-adhesive properties of ECM proteins were not commonly observed.

Background Information & Explanation
Laminins have multiple binding partners and also exert multiple biological functions by interacting with other ECM molecules and cell surface receptors. However, majority of the cell-binding sites map to regions distinct from the LCC domain. This domain is believed to play a key role in chain assembly yet it is considered a functionally silent domain, with a few exceptions.

Cell adhesion modulation is often related to cell migration. The actin cytoskeleton plays an important role in the processes of cell movement, with filopodia and lamellipodia formation being crucial. Because decreased actin stress fibre formation and increased membrane ruffle formation are consistent with the promotion of cell migration, it is a tempting speculation that the adhesive property of the LCC domain is linked to the modulation of cell migration in tissues where the LCC domain became accessible.


The Laminin Response in Inflammatory Bowel Disease: Protection or Malignancy? [6]

Study: When there is inflammation in Inflammatory Bowel Disease (IBD), mucosal ulceration and subsequent tissue repairs occur, promoting the constant remodelling of the BM, hence it is believed that laminin may play an unknown role in the inflammation response, impacting the bodies response to inflammation.

Figure 10 The nuclear accumulation of p53, triggered by inflammation, causes LMα1 to deposit onto the BM, accompanying LMα5. The increased expression of LMα1 and LMα5 in an inflamed microenvironment, potentially creates a physical barrier, resulting in attenuated inflammation, seen in the transgenic mice. Higher accumulation of this laminin in a carcinogenic environment though, is believed to contribute to pro - tumorigenic settings.

Furthermore, it has also been described that altered immunoreactivity of the BM constituents in the IBD, have shown an increase in alpha laminin, meaning it is likely that LM participates in the regeneration of the intestine.

Method: The above was highlighted when an increase in LMα1 and LMα5 expression was present in the colon tissues of IBD patients and in DSS-driven colitis in mice. The inflammation in these cases was accompanied by the nuclear accumulation of p53 as well as changes in the properties of the cell, caused in particular by the presence of the ulcer-associated cell lineage glands (UACL) in IBD. It was also revealed that the LMα1 and LMα5 chains were over-expressed, when inflamed, in human IBD and murine colitis specimens.

Results: The forced expression of LMα1 and LMα5 protected against DDS-induced inflammation, yet in carcinogenic conditions, accelerated colitis-associated tumorigenesis, as outlined in Figure 10. Furthermore, results suggested that LMs could play a role in tissue restitution, as LMs promoted wound closure in tissue rebuilding when there were disrupted epithelial cell monolayers.

Background Information & Explanation

Laminins are major components of the epithelial BM. They are also found present in intestinal BMs, functioning alongside collagen IV, perlecans and nidogens, as specialised ECM networks that separate epithelial cells from underlying connective tissue. Overall, they play a crucial role in tissue homeostasis and also have an important role in tissue maintenance and cancer progression, which represents an inherent risk of Irritable Bowel Disease (IBD).

Because the BM functions as a physical and chemical barrier, defects in the BM assembly or composition can cause several human disorders. Repetitive tissue destruction, repair and oxidative damage can together trigger mutagenesis and potentially initiate cancer causing events. In wound healing and tissue repair, it's made prevalent that inflammatory responses are commonly associated with the remodelling of the extracellular matrix (ECM). Altered ECM expression and ECM binding integrin adhesion receptors have been located in several inflamed tissues.


Keratinocyte – Targeted Expression of Human Laminin y2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice [2]

Study: To test whether or not the expression of the human laminin γ2 in mice helped prevent early lethality.

Method: A dox – controllable human laminin γ2 transgene was expressed, under a keratinocyte-specific promoter on the laminin γ2 knockout background.

Figure 11 The newborn knockout mice were noted to be smaller, have blistered feet and smaller pouches (B). The mice that carried both the transgenes (C), appeared very similar to the controls (A) (D). The rescued knockout mice survived to reach adulthood (F), with a similar length and weight to the wildtype littermates (E).

Results: Newborn offspring that lacked the transgene had blistered skin, resulting in a separted epidermal layer, smaller milk pouches and died a few days after birth. However, the mice that were carrying the human gene, as a result appeared similar to their wild-type littermates at birth, with similar weights and lengths, didn’t develop skin blisters, showing a more attached structure, were fertile and survived for more than 1.5 years. This comparison is shown in Figure 11.

Background Information & Explanation

Laminin-332 (LM-332) is heterotrimetric BM component comprised of α3, β3 and γ2 laminin chains, responsible for epithelial cell processes, including adhesion, migration and differentiation. It is present in the basement membranes of many embryonic and adult tissues, including the brain, gastrointestinal tract, heart, kidney, liver, lung, trachea, skin, spleen, thymus, salivary gland, mammary gland, ovary, prostate and testis.

Skin is a protective barrier, composed of two primary layers, known as the epidermis and dermis. These two layers are separated a sheet of specialised ECM known at the BMZ. The BMZ functions as a border, for structural support, influences cell attachment, proliferation, differentiation and migration. Should a defect occur in the structure or in one of the components of the BMZ, tissue separation and blister formation may result.

Keratinocytes are responsible for the secretion of LM-332 in the skin, which is identified as a key component of hemidesmosomes that connect the keratinocytes to the underlying dermis, functioning as an adhesion molecule between the epidermis and dermis. In mice, when any of the three LM-332 chains are not expressed, impaired anchorage and detachment of the epidermis occurs. This is similarly seen in human Junctional Epidermolysis Bullosa (JEB).

JEB is a genetic skin blistering disease, which results in, in the most severe cases, the death of infants within their first year of life. JEB is mostly caused by the absence of LM-332, due to mutations arising within the chains. Generally, those who experience skin blistering also experience blistering of the mucous membranes of the mouth and gastrointestinal tract, in turn affecting nutrition. This was confirmed on detection of laminin γ2 in the mouth of the mice, when examining the effects of the human gene expression on them.


Laminin – 511 A multi – functional adhesion protein regulating cell migration, tumor invasion and metastasis [7]

Study: To reinforce recent studies that indicate that Laminin-511 (LM-511) contributes to tumor dissemination and metastasis in advanced breast carcinomas as well as other tumor types. Furthermore, to look into experimental evidence that suggests that there is prognostic significance with higher expressions of LM-511 and that by targeting tumor-LM-511 interactions, there may be a therapeutic potential in advanced cancer patients.

Method: Specifically observing that metastatic breast tumor lines adhere and migrate more efficiently on LM-511, as opposed to on metastatic lines seen in the laboratory results are consistent with theories that metastatic breast tumors are mediated through attachments to LM-511. If observations are found correct then metastatic potential can be impacted by blocking the production of LM-511 and or the function and expression of its receptors.

Results: Increasing experimental evidence is supporting the link of LM-511 to cancer progression and reinforcing the belief that LM-511 has a broad role in tumor invasion and metastasis. However, questions in regards to its prognostic significance, precise mechanism of action and potential as a therapeutic target still need to be addressed and answered. As metastasis is responsible for majority of cancer – related deaths and is a clinical challenge, the continuation of research of LMs has the potential to present several information and solutions to overcoming this disease, as well as assist in the development of alternative strategies to prevent and/ or delay metastatic progression.

Background Information & Explanation

Laminins are abundant extracellular matrix proteins, present predominately in BMs. There are approximately 15 isoforms, named according to their specific combination of the α, β and γ chains. A combination of technological advances and experiments has contributed to the belief that LM-511 and its receptors regulate cancer progression.

The LM – 511 expression patterns vary between different tumor types whether it be basement membrane localisation to diffuse stromal or tumor cell expression. It’s level in tumor cells or surrounding vasculature varies based on the stage of tumor progression. Many studies show that LM-511 expressions in advanced tumors is maintained or even increased. It’s also observed that in advanced human breast cancers and bone metastases, there is high tumor cell expression of LM-511. Discussions are about whether future studies should look at LM-511 expression in tumors and whether it has a particular clinical relevance with a propensity to metastasise to bone. Whether or not LM-511 expression has any contribution to breast tumors, has not yet been fully confirmed.

As has been found from previous studies, many tumor lines synthesise, secrete and adhere to LM-511, potentially indicating that LM-511 is able to produce it’s effect on tumors via autocrine stimulation in part. Receptor studies are the reason for the many believed functions of LMs in breast cancer progression and metastasis and it's possibly inferred, from several observations that LM-binding integrins in metastatic breast tumors are mediated through attachment to LM-511.

A proposed model of LM-511 expression and function during cancer and metastasis has been developed, as outlined in Figure 12, incorporating current and previous findings.

Figure 12 (1) The tissue organisation is disrupted as a result of the loss of LM - 111 and LM - 332, uncontrolled growth of the tumor and continued expression of LM - 511, exposing the tumor to the surrounding stroma. (2) All the tumor cells undergo the epithelial to mesenchymal transition, under the influence of stromal factors, losing cell - cell contacts. (3) Tumor cell attachment and intravasation is supported by the increased LM - 511 amounts and overall changes in tumor vasculature LM isoforms. (4) Invasion of tumor cells into metastatic sites is supported through integrin and laminin interaction. When these integrins bind with laminin, tumor cells survive and grow.

Because there are no definitive answers in regards to the prognostic significance of LM-511, exact mechanism and potential therapeutically, the following should be looked into:

- For staining archival material, the generation of more robust antibodies.

- Clarification of the precise contribution of LM-511 to the metastatic process.

- Contribution (if any) of stromal/ vascular LM-511 to metastasis (tumor-derived LM-511 in metastasis is already supported).

- Studies in vivo, to provide proof of the therapeutic benefits that targeting LM receptors could have.


Remodeling of ECM by normal and tumor-associated fibroblasts promotes cervical cancer progression [74]

Study: The experiment was conducted to study the communication between tumor cells and fibroblasts.

Method: Light was shown through in - vitro models of tumor-stroma interaction. Some of the fibroblasts from the normal cervix and cervical cancers were grown separately, while others were in co-culture with CSCC7 cervical cancer cell-line. Changes in the glycoproteins, integrins and matrix metallo - proteases were examined.

Results: It was concluded that the tumor microenvironment played an influential role in the behaviour of cancer, with changes seen in the upregulation and redistribution of LM-111 in those cells which had undergone cancerous transformation, depositing into the BM and fibrillar connective tissue. It was also seen that there was a presence of activated myofibroblasts, which produce ECM, in the stroma. LM-5 was also found to reside in the cytoplasm of cancer cells, consistent with earlier findings. As a result, it was concluded that the tumor microenvironment played a pivotal role in the behaviour of cancer.

Background Information & Explanation

Tumor microenvironments have become the focus of intensive research for cancer therapy. Parenchymal cells and their stromal components are separated by a BM in normal epithelium. When a local host stroma is activated, the normal epithelium transitions into an invasive carcinoma. This malignant progression then prevents the normal functioning of the BM, causing the reorientation of its structure. As a result, the invasive tumor cells acquire a more metastatic phenotype, losing their epithelial characteristics. During this process, stromal cells release numerous microenvironment-influencing macromolecules, causing changes to the ECM.

Fibroblasts, which play a prominent role in the pathology of solid tumors, are characteristic cell types in the microenvironment. The cancer – associated fibroblasts exhibit large quantities of ECM protein, proteases, within the reactive stroma. Matrix metalloproteinases (MMP’s) degrade the BM and stromal ECM, influencing the production of malignant tumors. As a result, newly synthesised ECM proteins assist in the movement of motile tumor cells and the development of new vessels.

In order to display an invasive phenotype, cervical carcinomas use the stromal MMP’s. MMP-7 and MMP-9 are induced into the cancer cells, made larger by tumor - stromal interactions. The stromal cells generate growth signals to the cancer cells by integrins, which are essential for the communication between cancer cells and tumorous stroma. It is this integrin communication that plays a big role in the cells survival, proliferation, migration and tumor invasion.

The cytokine, TGF-β1, produced by fibroblasts is an important regulator of the ECM assembling and remodelling the ECM during cancer progression. Its known to exert a growth inhibitory action on epithelial cells, however this is lost in malignant transformation. This causes the growth factor of the ECM to convert from inhibitor to tumor promotor. This cytokine is activated through EC mechanisms such as proteases, thrombospodin – 1 and integrins. This exchange between tumor cells and fibroblasts is what influences growth factors, modifying the role they play on the tumor tissue.


Immunohistochemical analysis of laminin expression in adenoid cystic carcinoma [75]

Study: To understand the distribution and role of laminin within tumors by observing expression in adenoid cystic carcinomas (ACC) of the salivary gland origin, via immunohistochemistry.

Method: Polyclonal antihuman laminin primary antibodies were used on thirty cases of ACC. Specifically, intracellular, pseudocystic space inner borders, pseudocyst lumens, tumor islands and intervening stroma were observed for the distribution of laminin within the ACCs.

Figure 13 Distribution of Laminin in the Adenoid Cystic Carcinoma

Results: Of the 30 cases, approximately 15 showed the presence of laminin on the inner aspect of the pseudocystic spaces, 22 in the lumen, 20 within the intervening stroma, 16 bordering the tumor islands and 4 intracellular. See Figure 13. As a result, the conclusion that laminin plays a role in proliferation of tumor cells and in pseudocyst formation was reached, with further findings expressing the potential of laminin to contribute to the growth and differentiation of ACC and also assist in the assessment of the prognosis of tumors.

Background Information & Explanation

The epithelial malignant neoplasm of salivary glands is adenoid cystic carcinoma (ACC), a rare tumor accounting for approximately 7.5% - 10% of salivary gland neoplasms. It is slow growing but harmful, known to have a high recurrence rate and development of distant metastasis, even after treatment.

Laminin receptor overexpression on tumor cells at distant sites from primary lesions coupled with laminins ability to influence type IV collagenase production is likely to support the breakdown and perforation of the basement membrane by metastatic tumor cells. Therefore, it can be speculated that the growth and differentiation of ACC, could be influenced by the location of the laminin within. This in turn may contribute to understanding if the varying degrees of tumor aggression are related to location.

Laminins are known for their organisation role of the BM, and with increasing research, are believed to regulate cell proliferation, differentiation and function via their interactions with other surface molecules. Therefore, there is reason to believe that laminin does contribute to ACC growth and proliferation.

Excessive secretions of the ECM produced by tumor cells, based on morphological findings, have said to result in pseudocystic structures. BM rich matrix is believed to be crucial for the proliferation of tumor cells, because of the retention of the ECM within pseudocysts.

The molecules proliferate the cells as strands to communicate with the laminin molecule, before growing in various directions to contact each other. The stroma is divided, with the component enclosed by the tumor forming pseudocystic spaces.

Progression of tumor cells may give rise to solid growth patterns if they proliferate into the pseudocysts. ACC attachment to BM emphasises that tumor cells use the structure to invade, proliferate and travel, and when becoming more aggressive, cause a reduction in laminin expression.

Additional Information on Antibodies

Laminin antibodies are most predominantly used to highlight basement membranes and blood vessels.

Antibody
Species
Working Concentration
Secondary Antibody
Publication
Laminin Antibody 0.1 ml Basement Membrane Marker (NB300 - 144) Rabbit, polyclonal to Laminin 1 ug at 1.0 mg/ ml Anti - Rabbit IgG (H&L) (Goat) Antibody Peroxidase Conjugated <pubmed> 22511849 </pubmed>
Anti-Laminin 5 Antibody Rabbit, polyclonal to Laminin 5 50 µg at 1 mg/ml F(ab')2 fragment goat anti-rabbit Alexa Fluor 594 <pubmed>25133673</pubmed>
Laminin α-1 Antibody (M-20) Goat polyclonal IgG 200 µg/ml Donkey anti-goat IgG-HRP <pubmed>17364662</pubmed>
Anti-Laminin Antibody (ab11575) Rabbit polyclonal to Laminin 250 µl at 0.54 - 0.72 mg/ml Biotin - conjugated goat anti-rabbit IgG polyclonal (1/1000) <pubmed>24334445</pubmed>


Ultrastructural immunolocalization of laminin 332 (laminin 5) at dento-gingival interface in Macaca fuscata monkey [76]

Study: To investigate the dento – gingival interface of the Japanese monkey (Macaca fuscata) in order to examine the distribution of laminin – 332 (LM – 332), as well as identify the origin of the dental cuticle and supplementary lamina densa. Furthermore, to look into the precise localization of LM – 332 in the internal basal lamina.

Method: Investigation of the dento – gingival interface via pre – and post – embedding immunoelectron microscopy. Localization examined immunocytochemically by post – embedding high resolution immunogold labelling.

Results: On the inner basal lamina of the junctional epithelium (particularly on the lamina densa), laminin – 332 was detected. It was also noticed to appear scattered but specifically distributed on the dental cuticle, believed to be a secretory product of junctional epithelial cells.

Background Information & Explanation
The lamina densa and the lamina lucida compose the internal basal lamina. The lamina densa is much thicker then other BMs in the body. Part of it is known as the supplementary lamina densa.

The internal basal lamina (particularly the supplementary lamina densa) and hemidesmosomes at the dento – gingival border, help maintain the firm adhesion between the junctional epithelium of the gingiva and the tooth enamel.

Using the high-resolution electron microscopy, it was seen that the monkey tooth’s fine network structure was very similar to that of the lamina densa. However, specific origins of the dental cuticle components are yet to be identified, despite earlier findings suggesting that they are epithelial cell products.

Previous studies had revealed that the internal basal lamina contains LM – 332. This was consistent in this experiment. Studies also highlighted that gold particles were mainly situated on the lamina densa and minimally on the lamina lucida component that faces the lamina densa, which was also seen in this experiment. It was noticed that the particles gradually increased in number when moving from the apical toward the coronal portion on the internal basal lamina, emphasising that the lamina densa contains mainly LM – 332.

The possibility that junctional epithelial cells are the producers of LM – 332, was also noted, as tooth – enamel – facing cells strongly expressed LM – 332, with the expression gradually increasing from the cemento – enamel junction area to the gingival sulcus. Furthermore, the dental cuticle was specifically labelled by the immunogold particles, emphasising that the dental cuticle contains LM – 332.

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References

  1. 1.0 1.1 <pubmed>22319142</pubmed>
  2. 2.0 2.1 <pubmed>23029085</pubmed>
  3. 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 <pubmed>23076216</pubmed>
  4. 4.0 4.1 4.2 4.3 4.4 4.5 4.6 <pubmed>23263632</pubmed>
  5. 5.0 5.1 <pubmed>25310607</pubmed>
  6. 6.0 6.1 6.2 <pubmed>25347196</pubmed>
  7. 7.0 7.1 7.2 7.3 <pubmed>23076212</pubmed>
  8. <pubmed>25392494</pubmed>
  9. 9.0 9.1 9.2 <pubmed>22723936</pubmed>
  10. <pubmed>16505007</pubmed>
  11. <pubmed>24550734</pubmed>
  12. 12.0 12.1 12.2 12.3 <pubmed>12609502</pubmed>
  13. <pubmed>24830794</pubmed>
  14. Juan Fernando Maguid, 05/03/2011, An introduction to Laminins (animated movie), https://www.youtube.com/watch?v=QGnFgDEWCLs
  15. 15.0 15.1 <pubmed>114518</pubmed> Cite error: Invalid <ref> tag; name "PMID114518" defined multiple times with different content
  16. <pubmed>15979864</pubmed>
  17. <pubmed>24443019</pubmed>
  18. <pubmed>2534046</pubmed>
  19. <pubmed>6795355</pubmed>
  20. <pubmed>2404817</pubmed>
  21. 21.0 21.1 21.2 21.3 21.4 21.5 <pubmed>24706882</pubmed>
  22. 22.0 22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9 <pubmed>25015639</pubmed>
  23. 23.0 23.1 <pubmed>23209340</pubmed>
  24. <pubmed>25364175</pubmed>
  25. 25.0 25.1 <pubmed>19895795</pubmed>
  26. 26.0 26.1 26.2 26.3 26.4 26.5 26.6 26.7 <pubmed>24009313</pubmed>
  27. 27.0 27.1 27.2 <pubmed>23756649</pubmed>
  28. 28.0 28.1 28.2 <pubmed>19213874</pubmed>
  29. <pubmed>20566382</pubmed>
  30. <pubmed>8978824</pubmed>
  31. 31.0 31.1 31.2 <pubmed>24951591</pubmed>
  32. <pubmed>16714422</pubmed>
  33. <pubmed>8601594</pubmed>
  34. 34.0 34.1 34.2 34.3 34.4 <pubmed>16374451</pubmed>
  35. <pubmed>16672690</pubmed>
  36. 36.0 36.1 36.2 36.3 <pubmed>20211547</pubmed>
  37. 37.0 37.1 37.2 37.3 37.4 <pubmed>12743034</pubmed>
  38. <pubmed>21519194</pubmed>
  39. <pubmed>21766463</pubmed>
  40. <pubmed>18644586</pubmed>
  41. 41.0 41.1 41.2 <pubmed>21349841</pubmed>
  42. 42.0 42.1 42.2 42.3 <pubmed>15205311</pubmed>
  43. 43.0 43.1 43.2 43.3 <pubmed>21067603</pubmed>
  44. 44.0 44.1 44.2 44.3 <pubmed>16957169</pubmed>
  45. <pubmed> 11373318</pubmed>
  46. <pubmed>23263634</pubmed>
  47. <pubmed>22427740</pubmed>
  48. <pubmed>17457303</pubmed>
  49. <pubmed>21896617</pubmed>
  50. <pubmed>24497408</pubmed>
  51. <pubmed> 16441418</pubmed>
  52. <pubmed> 17533363</pubmed>
  53. <pubmed>21172822</pubmed>
  54. <pubmed>15024036</pubmed>
  55. <pubmed>23124251</pubmed>
  56. <pubmed>24118289</pubmed>
  57. Patricia G Greciano, Jose V Moyano, Mary M Buschmann, Jun Tang, Yue Lu, Jean Rudnicki, Aki Manninen, Karl S Matlin Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells. Mol. Biol. Cell: 2012, 23(1);121-36 PMID:22031290, https://www.youtube.com/watch?v=5Vg-dzM5tnU
  58. 58.0 58.1 58.2 <pubmed>23739692</pubmed> Cite error: Invalid <ref> tag; name "PMID 19330236" defined multiple times with different content Cite error: Invalid <ref> tag; name "PMID 19330236" defined multiple times with different content
  59. <pubmed>10367729</pubmed>
  60. <pubmed>11810295</pubmed>
  61. <pubmed>20301304</pubmed>
  62. <pubmed>23154401</pubmed>
  63. <pubmed>11938437</pubmed>
  64. <pubmed>11054875</pubmed>
  65. <pubmed>10022829</pubmed>
  66. <pubmed>16897576</pubmed>
  67. <pubmed>25753421</pubmed>
  68. <pubmed>15102706</pubmed>
  69. <pubmed>9885251</pubmed>
  70. <pubmed>23076210</pubmed>
  71. <pubmed>9852162</pubmed>
  72. 72.0 72.1 <pubmed>11809810</pubmed>
  73. <pubmed>23076210</pubmed>
  74. <pubmed>25885552</pubmed>
  75. <pubmed>PMC4211234</pubmed>
  76. <pubmed>25055992</pubmed>