Connective Tissue Histology 2022-2023 PDF

COURSE SUPPORT Histology, second year, Medicine – 2022-2023 Course 3 – CONNECTIVE TISSUE Histologic structure: (i) extracellular matrix, formed by an amorphous component and a fibrillar component (fibers of the connective tissue) (ii) cells: proper (fixed) and migrate...

COURSE SUPPORT Histology, second year, Medicine – 2022-2023 Course 3 – CONNECTIVE TISSUE Histologic structure: (i) extracellular matrix, formed by an amorphous component and a fibrillar component (fibers of the connective tissue) (ii) cells: proper (fixed) and migrated (free). Morpho-functional characteristics (1) mesenchymal origin (mesoderm and ectomesenchyme), (2) production of a large quantity of extracellular macromolecules that form a complex matrix (3) fibrillar organisation of some macromolecules (4) cell dispersion in matrix and, as a result, the absence of intercellular junctions (5) adhesion of cells with extracellular matrix, resulting a cell-matrix interaction This tissue contains multiple tissular types, which are distinct regarding cell population and extracellular material. Based on characteristics mentioned above, diverse classifications were proposed. Considering the didactic aspects, the following types of connective tissue may be mentioned: connective tissue proper (loose and dense connective tissues), connective tissue with special properties, and specialized connective tissue. This classification has been replaced by the following one: embryonic, proper, and specialized connective tissues, with the previous mention as separate categories of specialized connective tissue, the elastic and reticular tissues. Histogenetically, connective tissue derived from embrionar mesenchyme. 1. COMPONENTS OF THE CONNECTIVE TISSUE 1.1. EXTRACELLULAR MATRIX Extracelular matrix is the result of the synthesis of the proper cells of the connective tissue. It is under a permanently renewal process, through production and degradation of constitutive molecules, processes being in a perfect balance in normal conditions. Extracellular matrix is composed of an amorphous matrix and fibrilar matrix (fibrilar organisation). 1.1.1. AMORPHOUS MATRIX Amorphous matrix, also called ground substance or non fibrilar matrix, presented in vivo as a translucid material, is a highly hydrated gel, formed by a coloidal system dispersed in an aqueous phase. Coloidal system consists of macromolecules like glycosaminoglycans, proteoglycans and multiadhesive glycoproteins. 1.1.1.1. GLYCOSAMINOGLYCANS Most of the molecules present in the amorphous matrix are glycosaminoglycans (mucopolisaccharides), linear polymers formed by repetitive disaccharidic subunits. In a disaccharidic unit always exist a hexosamine (N-acetil-glucosamine/galactosamine) and an uronic acid (glucuronic/ioduronic). Glycosaminoglycans are of two types: nonsulfated (hyaluronic acid) and sulfated (keratan sulfate, heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate). 1.1.1.2. PROTEOGLYCANS Sulfated glycosaminoglycans are usually covalently bond with proteic molecules, to form proteoglycans. The aspect of proteoglycans is compared with a brush, in which the proteic core is the rod, while three-dimensional distributed glycosaminoglycans are the brush spikes. Hyaluronic acid does not form covalent bindings with proteic molecules, to form proteoglycans. Instead, through some specific connective molecules, the already formed proteoglycans can indirectly bind to hyaluronic acid, forming giant macromolecules, called aggregated proteoglycans– aggrecan compound. 1 Proteoglycans are classified in two types: extracellular and associated to the surface of the cell. Some of the most frequent proteoglycans are: decorin, sindecan, versican, fibromodulin, biglican, perlecan, and CD44. 1.1.1.3. EXTRACELLULAR GLYCOPROTEINS(STRUCTURAL) -are molecules with multiple functions in formation and maintaining of the molecular organization of the extracellular matrix and in establishing correlations between matrix and the cells. For this reason, they were also called multiadhesive glycoproteins. The most common multiadhesive glycoproteins are: fibronectin, laminin, entactin, fibrilin, tenascin, and trombospondin. 1.1.2. FIBRILLAR MATRIX The extracellular molecules have a particular organization, leading to the structuring of fibrillar matrix (fibers of the connective tissue). From a morphologic point of vue and based on the tinctorial properties, there have been described three types of fibers: collagen, reticular, and elastic. From a chemical point of vue, the three types of fibers result from the organization of two different molecules: (i) collagen, present in collagen fibers and in reticular fibers and (ii) elastin, present in elastic fibers. 1.1.2.1. COLLAGEN FIBERS Collagen represents a family of tough, inelastic, fibrillar scleroproteins, of a glycoproteic nature. The multiple existent types of collagen (more than 15) represent aproximately 20% from the proteins of the body. Light microscopy (LM) - routine HE staining: organized in bundless with diameters of 0.5-10 μm and indefinite length, acidophilic; wavy path, do not branch and do not anastomose; they pass from one bundle to another. Their orientation varies according to the type of connective tissue. - special stainings: acid fuchsine (red), aniline derivatives (blue), and light green (green). Usually, the use of these stainings is achieved by different techniques: van Gieson, Mallory trichrome, and Masson trichrome. - in polarized light, fibers: birefringent, which explains the existence of submicroscopic units, with parallel distribution to the long axis of the fiber. Electron microscopy (EM) - fibrills (fibrillar units) of 20-100 nm, with a parallel arrangement: - fibril: o cross section: rounded structure o longitudinal section: cylindrical, shows an axial periodicity formed by the alternance of electron-dense and electron-lucent bands, that repeat every 68 nm along the length of the fibril. Structure and molecular organization fibrillar unit is the result of the polymerisation of collagen molecules(tropocollagen). each fibril consists of a well organized assembly of 5 molecular subunits (microfibrils), each with 280-300 nm length and 15 nm diameter. 1.1.2.2. TYPES OF COLLAGEN - 19 types of classic collagen - 10 types of proteins containing in their structure domains with typical collagenic organisation. In each type of collagen there is the possibility that the three α chains to be identically or different. Types of collagen can be grouped based on criteria of organization (morphological, chemical etc.) in large categories: fibrillar collagen, fibril-associated collagens with interrupted triple helixes (FACITs), hexagonal network-forming collagens, transmembrane collagens, multiplexins, and basement membrane-forming collagens. 2 1.1.2.3. ELASTIC FIBERS Elastic fibers represent another component of the extracellular fibrilar matrix, in which the predominent molecule is elastin. LM: - special stainings (orcein, resorcin-fuchsin, and aldehyde-fuchsin) - individualized fine, curly fibers (1-3 μm), that do not form fascicles and tend to branch and anastomose, resulting networks with large mesh - fenestrated lamellae (the walls of the elastic arteries) of different dimensions, disposed in concentric manner = elastic laminae - localization: pulmonary alveoli, ligament flava associated with the spinal column, aortic tunica media EM - amorphous material with moderate electron density– pars amorpha - thin homogenous microfibrils, without striations, with a diameter of 10-12 nm, situated at the periphery of the amorphous substance Structure and molecular organization -2 proteic components, different from chemical and morphologic point of view: elastin (core) and fibrilin (periphery) 1.1.2.4. FIBRELE RETICULARE - composed of type III collagen - form a suuport network for cellular component of different tissues and organs - are produced by fibroblasts, with the exception of reticular fibers of endoneurium, which are produced by Schwann cells, reticular fibers of the blood vassels and digestive tube, where they are produced by smooth muscle cells LM - do not stain in routine H&E staining - may be identified by special stainings, showing thin and delicately woven strands - may be stained with PAS, silver staining – argyrophilic fibers stained in black (Gomori and Wilder staining) Locations: loose connective tissue at the limit between connective and epithelial tissues, around adipocytes, of blood vessels, of nerves and muscle cells, in embryonic tissues, in connective stroma of hemato-lymphopoetic organs (excepting the thymus) 1.2. CELLS OF THE CONNECTIVE TISSUE Regarding the embrionic origin, in the connective tissue there are two types of cells: proper and migrated. According to their stability cells may be: resident (fibroblasts, myofibroblasts, macrophages, adipocytes, mast cells, and adult stem cells) and wandering (lymphocytes, plasma cells, neutrophils, eosinophils, basophils, and monocytes) 1.2.1. PROPER CELLS Reprezented by mesenchymal cells, adult stem cells, fibroblasts/fibrocytes, and adipocytes (mesenchymal origin). 1.2.1.1. MESENCHYMAL CELL - specific to embrionic life -stelate or elongated shape, large ovalary or elongated pale nuclei, dispersed chromatin, with abundant cytoplasm, numerous cell processes, poor in organelles - are differentiated in proper cells of the connective tissue, endothelial cells, and smooth muscle fibers - only a few remain in a nondifferentiated stage, as pluripotent cells (remaining embrionic mesenchyme); associated to capillaries (pericytes) 3 1.2.1.2. ADULT STEM CELL -resides in specific sites called niches -niches of adult stem cells are located in different tissue and organs -compared with embryonic stem cells, adult stem cells cannot differentiate into multiple lineages, as they are capable of differentiation only on a single specific lineage -are present in stomach, small and large intestine 1.2.1.3. FIBROBLAST – FIBROCYTE Fibroblast – present in all types of connective tissue, derived from nondifferentiated mesenchymal cell, responsible of synthesis and degrdation of extracellular matrix Fibrocyte – fibroblast in a more diminished active synthesis stage Fibroblast and fibrocyte are the same cell in different functional stages. Fibroblast LM - elongated, fusiform or stelate cell, with a diameter between 20-30 μm / 5-10 μm - basophilic cytoplasm, with irregular processes - large, central, round or ovoid hypocromatic nucleus, granular chromatine, peripherically disposed, an evident nucleolus EM - active cell, specialised in proteic synthesis - all cell organelles: remarkable presence of rER (often distended because of its content), ribosomes, proeminent Golgi complex, sER, elongated mitochondria, with evident cristae Fibrocyte LM - filiform, elongated cell - eosinophilic cytoplasm - rod-shaped, elongated nucleus, condensed chromatin, without evident nucleolus EM - reduced rER - abundant free ribosomes - only a few mitochondria - less developed Golgi complex - lysosomes - a few elements of cytoskeleton. Functions - produce extracellular matrix and maintain the balance between synthesis and lysis - have receptors for LDL (proteins involved in atheromatosis genesis); through endocytosis mediated by receptors, the fibroblast catabolizes LDL, performing also a transfer of cholesterol on HDL (proteins non involved in atheromatosis) - produce interferons (IFNβ), substances with important role in natural defence - produce chemotactic factors, involved in nonspecific defence Myofibroblast -display properties of both fibroblasts and smooth muscle cells -is an elongated spindly connective tissue cell, not readily identifiable in routine H&E staining -is characterized by the presence of bundles of actin filaments, associated with motor proteins, such as nonmuscle myosin -expression of alpha-smooth muscle actin, specific for smooth muscle fiber, is regulated byTGF-beta1 -actin bundles cross the cell cytoplasm, attaching on the opposite sites of the inner plasma membrane -the site of actin fibers attachement to the plasma membrane serves as a cell-to-extracellular matrix (ECM) anchoring junction, called fibronexus 4  -this arrangement is the basis of a mechanotransduction system, in which the force generated by contraction of the intracellular actin bundles is transmitted to the ECM. EM - rER and Golgi complex - longitudinally disposed actin filaments - dense bodies similar to those observed in smooth muscle cells - as in the smooth muscle cell, the nucleus often has an undulating surface profile (aspect associated with cell contraction) - the absence of surrounding basal lamina makes it different from smooth muscle cell 1.2.1.4. ADIPOCYTE - differentiated cell for storage and release of lipids (especially tryglicerids) - during embrionic development: adipoblasts, from which result two types of cells: white adipocyte and brown adipocyte. White adipocyte (unilocular) - isolated or in small groups - in loose connective tissue - important clusters: form a particular type– adipose tissue LM - spherical cell (round on section), with a diameter of 80-100 μm - periphery: small cytoplasmic eosinophilic halo (1-2 μm thick) - central: large lipidic inclusion, resulted due to the coalescence of small inclusions (especially tryglicerids) - nucleus pushed to the periphery. In routine histologic techniques, due to the use of organic solvents, the lipidic inclusion dissapears and the cell appear empty. The nucleus being pushed to the periphery and the optical empty aspect determine the classic comparison of adipocyte with “signet ring”. To maintain the inclusion, freezing sections and the use of liposoluble stainings (Sudan III, IV, Scharlach, Nile blue etc.) are recommended. EM - lipidic inclusion is not delimited by a membrane - the limit between inclusion and cytoplasm: formed by two distinct components: a layer of condensed lipids (5 nm thick) surrounded by cellular cytoskeleton – microfilaments (diameter of 5 nm) with parallel disposition - perinuclear: Golgi complex, mitochondria, free ribosomes, reduced rER, microfilaments, and intermediate filaments - the rest of cytoplasm: sER, mitochondria, and elements of cytoskeleton Function - performs, in dinamic balance, lipogenesis (synthesis and storage of tryglicerids) and lipolysis. Brown adipocyte (multilocular) - caracteristic cell for hibernating animals, and in human, for the newborn LM - polyhedral cell, of 20-30 μm - spongy cytoplasm, due to the presence of numerous small lipidic inclusions, which do not confluence - spherical, usually excentrical nucleus EM - numerous spherical large mitochondria with abundant cytochrome oxidase which give the characteristic cells color - expresses a specific mitochrondrial protein called uncoupling protein (UCP-1) or thermogenin - cytoskeleton (explains the absence of confluence for the numerous lipidic inclusions) - β-adrenergic receptors in synaptic contact with autonomic simpatic fibers - reduced Golgi apparatus and small amount of rER and sER 5 Function - directly involved in thermogenesis. 1.2.2. MIGRATED CELLS Represented by macrophages, plasma cells (locally differentiated from T lymphocytes), lymphocytes, mast cells and, less numerous, eosinophiles. All these cells are very important in specific and non specific defence, especially in case of bacteria, and appear in zones invaded by microorganisms. 1.2.2.1. PLASMA CELL Plasma cells are in fact activated B lymphocytes as a result of an antigenic stimulation. LM - ovoid shape - dimensions of 20 μm - basophilic cytoplasm - eccentrically, round nucleus, with a perinuclear halo, central nucleolus, characteristic distribution of chromatin, as condensed clusters at the periphery, (heterochromatin alternating with zones of euchromatin); this aspect was compared with “cartwheel” or “clock face”. EM - microvilli - pseudopods - few centrioles surrounded by Golgi complex - well developed rER, ribosomes - few mitochondria Function - cell which synthesizes and secretes glycosylated polipeptides - their synthesis product is represented by immunoglobulins – plasma molecules with antibody activity (effectors of the immune humoral response). 1.2.2.2. MACROPGAGE Although it is a migrated cell, from a numerical point of view, in many types of connective tissue, macrophage is placed on second place after fibroblast. The origin of macrophages (also called hystiocytes in the connective tissue) is in circulating monocytes, which have the capacity that at some stimuli, to migrate from the vascular system in the connective tissue, where, after they become mature, have a life span of aproximately 60 days. Around 1970, van Fourth introduced the concept of Mononuclear Phagocyte System, which has three compartments: (i) central compartment, reprezented by the precursors of monocytopoiesis, localized in red bone marrow; (ii) distributive compartment, reprezented by circulating monocytes (with a life span of 72 hours in blood); (iii) tissular compartment, resulted from the migration of mature monocytes during circulating period, in different tissues. All the members of this system are born from stem cells from the red bone marrow, present lysosomes, are capable of phagocytosis and have receptors for Fc fragment of immunoglobulins and for conplement. Tissular compartment is represented by: − hystiocytes or macrophages of the connective tissue; − hepatic Kupffer cells; − macrophages of the lymphopoetic organs (spleen and lymph nodes); − pulmonary macrophages; − microglia of the nervous central system; 6 − dendritic epidermal cells – Langerhans cells; − osteoclasts of the bone. There are two different types of macrophages: resting and activated (by classical pathway – M1, which is involved in chronic inflammation and cellular lesions and by alternative pathway - M2, which is producing ECM components, such as fibronectin and othe adhesive glycoproteins and reduces inflammation). Another classification defines fixed macrophages, which exist in a connective tissue, and free macrophages, resulted as a result of the action of an exogenous stimulus which facilitate their migration in that place. The macrophages morphology is extremely diverse, because they are mobile cells, able to migrate from blood in the connective tissue and to a certain extent, to change their place in the connective tissue. LM - resting macrophage is difficile to differentiate from fibroblast - irregular, stelate or fusiforme shape, with a diameter of 10-30 μm - basophilic or acidophilic cytoplasm (in relation with the functional stage of the cell) - small, hyperchromatic central nucleus, frequently dispersed chromatin, nucleolus –sometimes present - activated macrophage – principal function: phagocytosis - unequal cell surface, with different processes (either short or filopodia, long, digitiform type), membranary folds - nucleus frequently indented on one side, reniform - in some locations, cytoplasm presents vacuoles and variate inclusions: lipidic material, endogenous pigments (bilirubin, hemoglobin, hemosiderin) or exogenous pigments (carbon particles, silica, etc.) EM – activated macrophage - surface cytoplasmic processes (indicator of phagocytosis) - small, dense, irregular and indented nucleus - well developed rER and sER - perinuclear, proeminent Golgi apparatus - mitochondria - cytoskeleton (actin microfilaments, microtubules, and intermediate filaments) - numerous small, dense granules, type membrane-bounded vesicles = lysosomes (indicator of phagocytosis) - endocytosis vesicles - secondary lysosomes. Principal functions: - phagocytosis - synthesis and secretion - Antigen presentation (role in immune response). 1.2.2.3. MAST CELL - derives from pluripotent hematopoietic stem cell, in red bone marrow, from a common precursor with the basophil LM - round or ovoid cell, diameter of 20-30 μm - central, spherical nucleus, relatively small comparatively with the cell dimension, with 1-2 evident nucleoli and granular chromatin - basophilic cytoplasm, filled with numerous intense basophil membrane-bounded granules, of 0.3-0.8 μm, which frequently mask the nucleus - difficult to identify in routine stainings, needs special stainings, because of its content of heparin (glycosaminoglycan sulfate – heparan sulfate), granules are metachromatic (the 7  colour changes from blue to red magenta) when using stainings based on derivatives of aniline. EM - reduced cellular organelles - dispersed rER - a few mitochondria - small Golgi complex - differences in shape, dimensions, and granular content, with variations in the same cell - cell membrane with folds and microvilli. Biochemical – granular content - primary or preformed mediators: heparin (anticoagulant factor, lipolytic), histamine, chondroitin-sulfate, neutral proteases (role in proteic cleavage for complement activation and consecutive in inflammatory response amplification), enzymes like tryptase and chymase, aryl sulfatase (inactivator for C leucotrienes, limiting the inflammatory response), β-glucuronidase, ECF, NCF, G catepsin - secondary or neoformed mediators: synthesis from precursors of membrane arachidonic acid: C4 and D4 leucotrienes and D2 prostaglandins Receptors of membrane surface: receptors with high affinity for IgE. Mast cell population: two different cell types, based on location and granular content: mast cells of the proper connective tissue and mucosal mast cells Functions - synthesis and storing of active biological products: preformed mediators and neoformed mediators (synthesized based on membranary phospholipids) - release through exocytosis 2. TYPES OF CONNECTIVE TISSUE Depending on its location, structure and multiple functions, connective tissue presents numerous varieties. Didactical classification: three principal types: - connective tissue proper - connective tissue with special properties - specialized connective tissue. Connective tissue proper includes, based on the existent raport between the constitutive elements (extracelular nonfibrilar matrix and fibrilar matrix and cells): - loose connective tissue - dense connective tissue - reticular connective tissue - elastic connective tissue - mucous connective tissue. Connective tissue with special properties is a heterogenous type, in which the cells are differentiated and specialised for some functions– for example, adipose tissue. Specialized connective tissue is present in two forms–cartilage and bone– the proper cells and the extracellular matrix are specialised for organism support, as part of the skeleton. 2.1. CONNECTIVE TISSUE PROPER - relative quantity of fibrilar, non fibrilar matrix and cells - the quantitative differences of their raport varies depending on their localisation, with a correlation between histoarchitecture and function. 2.1.1. LOOSE CONNECTIVE TISSUE - aproximately equal proportion of the three components: cells, fibers, and nonfibrilar extracellular matrix 8 - other names: areolar –by the way of fibriles arrangement, diffuse – by its large distribution and cellular –due to the presence in large number of both proper and migrated from blood vessels. Characteristics: - collagen, reticular and elastic fibers, dispersed or organised in a tridimensional network, determining the existance of some areolar spaces, which contain abundant ground substance and cells - collagen do not form thick bundles of fibers – fibers are individualised, forming irregular trajectories and have variate orientations - proper and migrated cells are present in different proportion, depending on the function of the tissue: prevail fibroblasts and macrophages, and in some areas, white adipocytes are also present, solitary or in small groups - presence of small nerous fibers and numerous capillaries– ensure hydroelectrolytic, gaseous, and nutritive exchanges, between blood and different cells. Localization - the most widespread type in human body - takes part to the formation of stroma of different organs (sometimes as septae that delimitate lobules) - through the basement membrane comes in contact with lining epithelial tissue, forming the mucosae chorion or lamina propria (digestive and respiratory system), having numerous cells involved in defence (mast cells, macrophages, plasma cells, and lymphocytes), but also numerous capillaries, and the superficial dermis (papillary) - comprise serosa which form the largest natural cavities (peritoneum, pleura, and pericardiums), and soft meninges - forms connective tissue sheats around nervous fibers and external tunica (adventitia) of the blood vessels 2.1.2. DENSE CONNECTIVE TISSUE - predominance of the fibers of the connective tissue, frequently arranged in bundles - two subtypes: - dense irregular connective tissue (bundles of fibers with different orientations) - dense regular connective tissue (bundles of fibers with parallel orientation to each other) 2.1.2.1. DENSE IRREGULAR CONNECTIVE TISSUE - formed, mostly, from type I collagen, disposed in fibers forming thick fascicles, with a variate spatial orientation, among which there are dispersed networks of elastic fibers. Localization - profound dermis of the skin - connective capsules that surround some internal organs (spleen, testis, liver, kidney, lymph nodes) and, sometimes, septae that delimitate their internal part - dura mater - the sheats of the large nerves 2.1.2.2. DENSE REGULAR CONNECTIVE TISSUE - formed by thick collagen type I fascicles, densely packed and oriented on the direction of action of traction mechanical forces, to provide maximum strength - reduced non fibrilar extracellular matrix, few cells (fibrocytes, distributed between fibers, with long axis parallel with collagen fibers) Localization - resistant support and protection structures: - tendons, ligaments, - aponeurosis - cornea - capsules covering the organs 9  TENDON - cord-like structures, that attach muscle to the bone - consist of parallel bundles of collagen fibers - between these bundles are rows of fibroblasts called tendinocytes - tendinocytes-surrounded by a specialized ECM that separates them from collagen fibers - epitendineum-thin connective tissue capsule surrounding the tendon - endotendineum-connective tissue with small blood vessels and nerves, subdividing the tendon into fascicles. LM - in H&E stained cross sections, appear stellate - in H&E stained longitudinal sections, tendinocytes appear as rows of tipically flattened basophilic nuclei, the cytoplasmic processes extended from their cell body are not usually evident because they blend in with the collagen fibers. EM - in longitudinal section present cytoplasmic processes and appear flat, between collagen fibers. 2.1.3. RETICULAR CONNECTIVE TISSUE - predominance of reticular fibers (type III collagen) in a tridimensional newtwork with smaller or larger meshes Localization - stroma for hematopoietic and lymphoid organs - sustain the hepatic sinusoids, adipose tissue, smooth muscle, pancreatic Langerhans islets. 2.1.4. ELASTIC CONNECTIVE TISSUE - abundance of elastic fibers, or as elastic lamellae Fibrilar type: - individualized fibers, well represented between collagen fibers, sometimes forming fascicles. Localization: interalveolar pulmonary septa, lamina propria of the urinary bladder, the dermis of the skin. Lamellar type - parallel arranged fibers, in thin fenestrated membranes Localization: large blood vessels (tunica media, elastic type), yellow ligament of spine, and suspensory ligament of penis. 2.1.5. MUCOUS CONNECTIVE TISSUE - present in umbiical cord -specialised extracellular matrix, gelatin-like, compose mainly of hyaluronic acid -its ground substance is reffered to as Wharton jelly, occupies large intercellular apaces located between thin collagen fibers. -presents spindle-shaped cells widely separated, appear much as fibroblasts in the near- term umbilical cord (the cytoplasmic processes are thin and difficult to visualize in routine H&E staining. 2.2. CONNECTIVE TISSUES WITH SPECIAL PROPERTIES 2.2.1 WHITE ADIPOSE TISSUE -represents at least 10% of the body weight of a normal healthy individual -forms a fatty layer of the subcutaneous (superficial) fascia, called the paniculus adiposus in the connective tissue beneath the skin -localised as storage of fat tissue in the connective tissue under the skin of the abdomen, buttocks, axilla, and thigh -is preferentially located in greater omentum, mesentery, retroperitoneal space-around the kidney, bone marrow, and between other tissues -secretes a variety of adipokines, which include hormones, growth factors, and cytokines 10  -leptin, the most important member of adipokines, involved in the regulation of the energy homeostasis and it is exclusively secreted by adipocytes -adiponectin- stimulates the fatty acids oxidation in muscles and liver, decrease the concentration of tryglycerids and glucose in plasma, raises the cell sensitivity to insulin -adipophilin-specific marker for lipid accumulation in the cell -adipsin –serine proteinase that regulates adipose tissue metabolism, by facilitating fatty acid storage and stiulating triglyceride synthesis -angiotensinogen (AGE) and angiotensin II -apelin- increases cardiac muscle contractility, decrease blood pressure -insulin-like growthb factor (IGF-1) -plasminogen activator inhibitor 1 (PAI-1) -interleukin 6 (IL-6) -resistin -prostaglandins I2 and F2 alpha (PGI2 and PGF2alpha) - retinol binding protein 4 (RBP-4) - transforming growth factor beta (TGF-beta) - tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) -visfatin Functions -storage of energy -thermal insulation -mechanical protection of vital organs -hormone secretion 2.2.2. BROWN ADIPOSE TISSUE (multilocular) - abundant in newborn, markedly reduced in adults - located on the back, along the upper half of the spine and toward the shoulders - the amount of brown adipose tissue gradually decreases as the body grows, but it remains present during the first decade of life in the cervical, axillary, paravertebral, mediastinal, sternal, and abdominal regions - then disappears from most sites, except for regions around the kidney, adrenal glands, large vessels (aorta), and regions of the neck (deep cervical and supraclavicular), back (interscapular and paravertebral), and thorax (mediastinum). - the brown adipocytes are smaller than white adipocytes. 11