Extra Cellular Matrix (ECM)

Extra cellular matrix is recently being explored in connection with cancer , metastases and autoimmune disorders. It is prepared for the benefit of both UG and PG medical and dental students.

1. Extra Cellular Matrix(ECM ) Dr.S.Sethupathy,M.D.,Ph.D ., Professor of Biochemistry, Rajah Muthiah Medical College, Annamalai University .

2. ECM -Definition Extracellular matrix  ( ECM ) is a network of extracellular macromolecules, such as collagen, enzymes, and  glycoproteins , that provide structural and biochemical support to surrounding cells ECM  includes the interstitial matrix and the  basement membrane Gels of  polysaccharides  and fibrous proteins fill the  interstitial space  and act as a compression buffer against the stress placed on the ECM

3. Functions of ECM

4. Functions of ECM ECM provides support, segregate tissues from one another, and regulate intercellular communication.  It sequesters a wide range of cellular growth factors and acts as a local store for them. Changes in physiological conditions can trigger protease activities that cause local release of them rapidly. It is essential for  growth, wound healing and fibrosis

5. Functions of ECM The stiffness and elasticity of the ECM is implicated in cell migration, gene expression, and differentiation. Cells actively sense ECM rigidity and migrate preferentially towards stiffer surfaces in a phenomenon called  durotaxis . Cell-to-ECM adhesion is regulated by specific cell-surface  cellular adhesion molecules (CAM) known as  integrins . The attachment of fibronectin to the extracellular domain initiates intracellular signalling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules such as  actin

6. Functions of ECM Extracellular matrix has been found to cause regrowth and healing of tissue. ECM works with stem cells to grow and regrow all parts of fetuses that gets damaged in the womb. It prevents the immune system from triggering from the injury It facilitates the surrounding cells to repair the tissue instead of forming scar tissue

7. Medical applications of ECM ECM is extracted from  pig bladders used regularly to treat ulcers by closing the hole in the stomach ECM from pig small intestine is used to repair " atrial septal defects" (ASD), "patent foramen ovale " (PFO) and  inguinal hernia.  ECM proteins used in cell culture systems to maintain stem and precursor cells in an undifferentiated state ECM used to induce differentiation of epithelial, endothelial and smooth muscle cells in vitro.

8. ECM molecules

9. Basement membranes are sheet-like depositions of ECM on which various  epithelial  cells rest. Collagen  fibers and  bone mineral  comprise the ECM of  bone tissue. Reticular fibers and  ground substance  comprise the ECM of  loose connective tissue; Blood plasma  is the ECM of  blood. Some single-celled organisms adopt multicellular   biofilms  in which the cells are embedded in an ECM composed primarily of  extracellular polymeric substances (EPS)

10. GAGs Components of the ECM are produced intracellularly by resident cells and secreted into the ECM via  exocytosis . They then aggregate with the existing matrix. ECM is composed of an interlocking mesh of fibrous  proteins  and  glycosaminoglycans (GAGs). Glycosaminoglycans  (GAGs) are  attached to ECM proteins to form  proteoglycans  (Except hyaluronic acid).

11. ECM as a store of growth factors Proteoglycans have a net negative charge It attracts positively charged sodium ions (Na + ) This attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans help to trap and store growth factors within the ECM .

12. Heparan sulfate Heparan sulfate  ( HS) as a   proteoglycan   (PG) attached in close proximity to cell surface or ECM proteins. It regulates a wide variety of biological activities, including  developmental process, angiogenesis, blood coagulation and tumor metastasis. In ECM, to basement membrane proteins  such as agrin , perlecan and collagen XVIII , heparan sulfate is attached.

13. Chondroitin sulfates  contribute to the tensile strength of cartilage, tendons, ligaments and walls of the  aorta . They have also been known to affect  neuroplasticity Keratan sulfates  have a variable sulfate content and, unlike many other GAGs, do not contain  uronic acid . They are present in the  cornea, cartilage, bones and the  horns  of  animals .

14. Non- proteoglycan polysaccharide- Hyaluronic acid Hyaluronic acid resist compression by providing a counteracting  turgor  (swelling) force by absorbing significant amounts of water. It is found in abundance in the ECM of load-bearing joints. It regulates cell behavior during embryonic development, healing processes, inflammation, and tumor development. It interacts with a specific trans-membrane receptor, CD44

15. Collagen Collagen  is the main structural protein  in the extracellular matrix in the various  connective tissues  It is the most abundant protein in mammals-25% to 35% of the whole-body protein content. It is a  triple helix  of elongated fibril called   collagen helix. It is mostly found in fibrous tissues such as  tendons, ligaments, and skin . The  fibroblast  is the most common cell that produces collagen

16. Collagen amino acids Glycine  is found at almost every third residue . Proline   makes up about 17% of collagen. Hydroxyproline  derived from proline   Hydroxylysine derived from  lysine  - depending on the type of collagen, varying numbers of hydroxylysines are  glycosylated   Cortisol stimulates  degradation of (skin) collagen into amino acids.

17. Collagen types and functions 30 types of collagen are identified. The five most common types are: Type I-  skin, tendon vasculature, organs, bone (main component of the organic part of bone) Type II-  cartilage (main collagenous component of cartilage) Type III - reticulate (main component of reticular fibers), commonly found alongside type I Type IV : forms basal lamina, the epithelium-secreted layer of the basement membrane Type V : cell surfaces, hair, and placenta

18. Fibroblasts produce collagen fiber. Epithelial and smooth muscle cells produce type IV collagen Collagen

19. Collagen synthesis Inside the cell two types of alpha chains – alpha-1 and alpha 2 , are formed during translation on ribosomes along the rough endoplasmic reticulum (RER). These are preprocollagen having registration peptides one on each end and a signal peptide on N-terminal side. Polypeptide chains are released into the lumen of the RER. Signal peptides are cleaved inside the RER and the chains are now known as pro-alpha chains. Hydroxylation of  lysine  and  proline  amino acids occurs inside the lumen. This process is dependent on  ascorbic acid (vitamin C) as a  cofacto r, In  scurvy , the lack of hydroxylation of prolines and lysines causes a looser triple helix . Glycosylation of specific hydroxylysine residues occurs.

20. Collagen synthesis Now two alpha-1 chains and one alpha-2 chain form triple helical structure - procollagen inside the RER. Procollagen   is sent to Golgi apparatus , where it is packaged and secreted by  exocytosis . Outside the cell Registration peptides are cleaved and tropocollagen is formed by  procollagen peptidase . Multiple tropocollagen molecules form collagen fibrils, via covalent cross-linking ( aldol reaction ) by  lysyl oxidase ( copper dependent ) which links hydroxylysine and lysine residues. Multiple collagen fibrils form into collagen fibers . Collagen may be attached to cell membranes via several types of protein,   fibronectin ,  laminin ,  fibulin  and  integrin .

21. Collagen synthesis

22. Prolyl and lysyl hydroxylase – vitamin C

23. Vitamin C deficiency

24. Collagen Medical applications The collagenous   cardiac skeleton  - four  heart valve  rings, interventrivular septum, atrio ventricular septum are histologically , elastically and uniquely bound to cardiac muscle. The collagenous structure dividing the upper chambers of the heart from the lower chambers is an impermeable membrane that excludes both blood and electrical impulses Collagen has been widely used in cosmetic surgery, as a healing aid for burn patients, for reconstruction of bone and variety of dental, orthopedic, and surgical purposes.

25. Medical applications Collagen is used in bone grafting as it has a triple helical structure, making it a very strong molecule. Collagen scaffolds are used in tissue regeneration , as sponges, thin sheets, or gels. Collagen is used as a natural wound dressing . It is resistant against bacteria, It helps to keep the wound sterile , because of its natural ability to fight infection.  Collagen is used for  cell culture , studying cell behavior and cellular interactions with the  extracellular environment

26. Osteogenesis imperfecta (OI) OI is a group of genetic disorders that mainly affect the bones. - means imperfect bone formation Mutations in the  COL1A1  and  COL1A2  genes cause type I collagen defect Bones fracture easily, often from mild trauma or with no apparent cause. Multiple fractures are common, even before birth.  Other features are blue sclerae of the eyes, short stature, curvature of the spine (scoliosis), joint deformities (contractures), hearing loss, respiratory problems, and a disorder of tooth development called  dentinogenesis imperfecta . 

27. OI X ray –OI and Normal

28. Ehlers- Danlos syndrome A group of rare  genetic connective tissue disorders Symptoms include loose joints , joint pain, stretchy velvety skin, and abnormal scar formation .  At birth or in early childhood. Complications may include  aortic dissection,joint dislocations, scoliosis, chronic pain, or osteoarthritis. AR or AD manner Musculoskeletal symptoms include hyperflexible joints that are unstable and prone to sprain ,dislocation, subluxation , and hyperextension.

29. Ehlers- Danlos syndrome – Hyperflexible joints , laxity of skin

30. Alport syndrome It is a glomerular basement membrane disease  caused by a defect in collagen IV - Nephritis X-linked dominant commonly or recessive disorder ocular  features are corneal opacities, anterior lenticonus and cataract, central perimacular and peripheral coalescing fleck retinopathies, and temporal retinal thinning. Rarely a macular hole, or a maculopathy impairs vision Deafness Nephritis,Hematuria , later renal failure

31. Alport syndrome - Lenticonus , macular hole

32. Dystrophic Epidermolysis Bullosa (DEB) DEB is characterised by the site of blister formation in the lamina  densa  within the basement membrane zone and the upper dermis . It causes  generalised  blistering of the skin and internal mucous membranes  such as the  oesophagus , stomach and respiratory tract and leads to scar  formation DEB is due to mutationsin COL7A 1 gene encoding  collagen type VII, either AR or AD Blisters occur with minor trauma or friction and are painful. It can be mild to fatal and cause squamous cell cancer.

33. DEB

34. Chondrodysplasias Rare   autosomal recessive  Chondrodysplasia or acromesomelic dysplasia characterized by severe dwarfism at birth. Epiphyseal and metaphyseal abnormalities Abnormalities confined to limbs Severe shortening and deformity of long bones Fusion or absence of carpal and tarsal bones, ball shaped fingers Occasionally ,  polydactyly  and absent joints. Facial features and intelligence are normal . Type II collagen defect

35. Spondyloepiphyseal dysplasia (SED) is a group of disorders with primary involvement of the vertebrae and epiphyseal centers resulting in a short-trunk disproportionate dwarfism. Spondylo - refers to the spine, epiphyseal refers to the growing ends of bones, and dysplasia refers to abnormal growth .

36. Collagen vascular diseases Ankylosing spondylitis . Dermatomyositis . Polyarteritis nodosa . Psoriatic arthritis. Rheumatoid arthritis. Scleroderma. Systemic lupus erythematosus .

37. Collagen vascular diseases

38. Scleroderma  A group of  autoimmune diseases  affecting the  skin, blood vessels, muscles, and internal organs . The disease can be either localized to skin or involve other organs   There will be areas of thickened skin, stiffness, feeling tired, and poor blood flow to the fingers or toes with cold exposure . A form CREST syndrome classically results in  C alcium deposits,  R aynaud's syndrome,   E sophageal problems ,  S clerodactyly thickening of the skin of the fingers and toes T elengiectasias (areas of small dilated blood vessels .) An  abnormal immune response due to certain   genetic  factors, and exposure to  silica cause abnormal growth of  connective tissue  

39. Scleroderma- CREST syndrome

40. Scleroderma

41. Localized collagen defects Hypertrophic scar – excess collagen , raised scar Keloid – scar goes beyond wound boundaries does not regress Beal’s syndrome- contracture of hip, knee, elbow ankle joints Peyronie’s disease – deposition of abnormal type 1 and III collagen in the penis

42. Elastin It is highly  elastic  and present in  connective tissue  allowing many tissues to resume their shape after stretching or contracting. It helps skin to return to its original position when it is poked or pinched. ELN mutations are autosomal dominant . Abnormally long version of the tropoelastin protein interferes with the formation of mature  elastin  and the assembly of elastic fibers. It weakens connective tissue in the skin and causes cutis laxa . Elastin is encoded by the  ELN  gene.

43. Skin

44. Defective Elastin - Cutis laxa

45. Fibrillin  is a  glycoprotein , which is essential for the formation of  elastic fibers  found in  connective tissue . It is secreted into the  extracellular matrix  by  fibroblasts   Gets incorporated into the insoluble microfibrils , a scaffold for deposition of  elastin Fibrillin-1 is a major component of the  microfibrils  that form a sheath surrounding the amorphous  elastin . Marfan syndrome  is due to defective  FBN1  gene. Mutations in  FBN1  and  FBN2  are also associated with adolescent idiopathic  scoliosis

46. Fibrilin

47. Marfan syndrome (MFS) MFS is caused by a mutation in  FBN1 , that makes  fibrillin , results in abnormal connective tissue.    AR disorder  Patient tends to be tall and thin, with long arms, legs, fingers and toes. They also have  flexible joints and scoliosis.  An increased risk of  mitral valve prolapse  and  aortic aneurysm. The lungs, eyes, bones, and the covering of the spinal cord are also commonly affected

48. Marfan syndrome A positive wrist sign in a person with Marfan syndrome (the thumb and little finger overlap when grasping the wrist of the opposite hand) A positive thumb sign

49. Marfan syndrome

50. Syndecan

51. Fibronectin

52. Fibronectin

53. Laminin

54. Laminin

55. Laminin

56. Integrin

57. Integrin functions

58. Integrin

59. integrin

60. Clinical importance of ECM

61. T H A N K Y O U