In mice, lung formation begins at 9.5 days postcoitum (E9.5), when the foregut endoderm invades the splanchnic mesenchyme and undergoes dichotomous branching. Branching morphogenesis depends on mesenchymal–epithelial cell signaling using fibroblast (FGF10), hepatocyte (HGF), and transforming (TGFβ) growth factors, bone morphogenetic protein BMP4, sonic Hedgehog, and Wnt morphogen (Sect. 11.3.4.2). Factors EGF, FGF, and PDGF are involved in alveologenesis.

Fibroblast growth factors are important regulators of the respiratory tract genesis. Fibroblast growth factor-7 and -10 diffuse from the mesenchyme and activate alternatively splice variant IIIb (of extracellular loop III) of protein Tyr kinase receptor FGFR2IIIb in the nearby epithelium and induce budding that is composed of an inner endodermal epithelium and an outer mesenchyme. The expression pattern of specific FGFs at distinct steps of bud emergence controls regional endodermal cell proliferation.

Isotype FGF10 is produced by the mesenchyme at the earliest stages of lung-bud formation with 2 primordial buds from the trachea, from which the bronchial tree arises using a process of elongation, budding, and branching. Agent FGF10 stimulates proliferation of endodermal cells in the apical region of developing buds [1395].

Epidermal growth factor, transforming growth factor-α, and amphiregulin are produced by the mesenchyme and act via the common EGFR receptor [1396].

Hepatocyte growth factor is produced by the mesenchyme and its receptor is confined to the epithelium [1396]. Agent HGF supports action of other growth factors in branching genesis.

Concentrations of insulin-like growth factors IGF1 and IGF2 as well as that of their receptors do not change during gestation; IGF-binding proteins (IGFBP1–IGFBP6) are developmentally regulated [1396]. Receptor of IGF1 is necessary to avoid lung hypoplasia, but does not intervene in branching formation. Receptor of IGF2 is required for normal alveolus development.

Production of platelet-derived growth factor is restricted to the epithelium, whereas its cognate receptors localize to the mesenchyme [1396]. Homodimer PDGFaa contributes to branching regulation. Protomer PDGFa is required for alveolus septation.

Transforming growth factor-β1 to -3 as well as receptors TβR1 and TβR2 are involved in embryonic lungs. Agents TGFβ1 and TGFβ2 are produced by the mesenchyme and epithelium, respectively [1396]. They impede the generation of pulmonary branching via TβR2. On the other hand, TGFβ3 stimulates lung development.

Bone morphogenetic proteins intervene in lung development, in particular in the proximal–distal patterning. Isotype BMP4 is highly produced by epithelial cells of bud tips and, to a lesser extent, in the surrounding mesenchyme [1396]. It precludes epithelial cell proliferation.

Members of thenetrin andsemaphorin families participate in budding and branching during lung morphogenesis. Netrin-1 and -4 are produced by epithelial cells of bud stalks. They are deposited in the surrounding basement membrane, where they preclude ectopic budding and inappropriate lateral branching [1395]. Both netrin-1 and -4 are expressed at their highest levels at non-branching proximal endoderm and stalk region of growing buds. On the other hand, they are excluded from growing bud tips, where most of the branching occurs. The spatial restriction of netrins permits efficient branching genesis. In addition, netrin-4 and, to a lesser extent, netrin-1 inhibit the formation of secondary buds induced by FGF7 or FGF10 [1395]. Semaphorin-3A is expressed by the mesenchyme that surrounds the ends of buds. It organizes the size and shape of the emerging buds.

Wnt morphogens signal in progenitor cells of the respiratory epithelium both during development and after injury. Activation of β-catenin leads to its translocation to the nucleus and formation of a transcriptional activation complex with eitherT-cell factor (TCF),sex-determining region Y (SRY)-box gene product Sox9, orpituitary (or Paired-like) homeobox transcription factors Pitx1 and Pitx2 to cause the transcription of target genes, such as neuroblastoma-derived MYC (NMYC), Bmp4, and Fgfr2b in epithelial cells, andMYC, PITX2, and Fgfr2c in mesenchymal cells [1397]. Once bound to the mesenchymal fibroblast growth factorFGF10,FGFR2b receptor triggers the β-catenin pathway. In the mesenchyme, mesothelial and epithelial fibroblast growth factor FGF9 connects to FGFR2c to also initiate the β-catenin pathway. β-Catenin in the mesenchyme controls the differentiation of angioblasts into endothelial cells as well as proliferation of parabronchial smooth muscle progenitors [1397].

β-Catenin reduces the expression of epithelial differentiation markers during the early pseudoglandular stage of lung development [1397]. During early lung development, β-catenin signaling triggers the proliferation of epithelial differentiation marker-negative (surfactant protein SPc − and secretoglobin ScGb1a1 − )⁴ cells that may be precursors of SPc + , ScGb1a1 + cells. Very early SPc − progenitor cells localize to a slightly more proximal position than SPc + cells at the distal tip of the developing lung.

In adult lungs that undergo slow turnover and possess regenerative capacity via activation of endogenous stem cell populations, SPc + , ScGb1a1 + cells at the bronchoalveolar junction represent lung epithelial progenitor cells that can give rise to both alveolar and bronchiolar cell lineages, especially bronchiolar Clara cells. β-Catenin controls the balance between progenitor expansion and epithelial differentiation. During lung epithelium regeneration, the canonical Wnt pathway is activated in niches at the bronchoalveolar duct junctions that contain bronchoalveolar stem cells [1398].

Transcription factor GATA6 synthesized in the respiratory epithelium regulates the appearance timing and amount of bronchoalveolar stem cells. Its absence causes the precocious occurrence of bronchoalveolar stem cells and lack in epithelial differentiation. It activates the synthesis of the epithelial Wnt receptor Frizzled-2 [1398]. Non-canonicalFrizzled-2 receptor precludes early epithelial canonical Wnt–β-catenin signaling. Frizzled-2 also regulates effectors of non-canonical Wnt signaling, such as G-proteins andcalcium–calmodulin-dependent protein kinase CamK2. Moreover, GATA6 is needed for proper lung epithelial regeneration and differentiation of bronchoalveolar stem cells.

P38α mitogen-activated protein kinase activates transcription factors, such asCAAT/enhancer-binding protein C/EBPs and Forkhead box protein FoxA2 (a.k.a. hepatocyte nuclear factor HNF3β) that are necessary for the correct differentiation of the stem cells into alveolar type-2 and Clara cells [1399]. It controls self-renewal of the lung stem and progenitor cells, as it coordinates proliferation and differentiation signals.⁵ In particular, it impedes the action of mitogenic factors, especiallyepidermal growth factor receptor, hence restraining the proliferation of stem and progenitor cells.

Forkhead box protein FoxA2 operates in lung maturation and differentiation ofgoblet cells;FoxJ1 in proper development ofciliated cells; andFoxF1 in formation of pulmonary capillaries.

Forkhead box factor FoxM1 does not alter lung growth, in particular epithelial proliferation and airway morphogenesis, but prevents lung maturation and causes postnatal respiratory failure, as it is required for adaptation to air breathing [1400]. It is necessary foralveolus epithelial type-1 cell differentiation and synthesis of mucin-type type-1 integral membrane glycoproteinpodoplanin (Pdpn),⁶aquaporin-5, andsurfactant protein-A to -D. Moreover, it is a transcriptional activator of the genes that encode surfactant protein-A and -B. In adddition,FoxP2 contributes to postnatal lung alveolarization.

Many other transcription factors are produced in cells of the primitive lung, such asNKx2-1,GATA5 and GATA6,members of the C/EBP family,HoxA5 and HoxB3 to HoxB5, andnMyC, as well as intracellular (nuclear) receptors such as the glucocorticoid receptor (GR or NR3c1) [1396].

In the vasculature,vascular endothelial growth factor, especially VEGFa via VEGFR2, causes angiogenic sprouting. Tip cells emit numerous filopodia and migrate in a given direction under the guidance of VEGFa (Chap. 10). Whereas tip cell migration depends on a gradient of VEGFa, the followers — stalk cells — proliferate under the control of VEGFa concentration [1394]. Selection of tip from stalk cells results from Delta-like ligand DLL4 and its receptorNotch-1. Ligand DLL4 is preferentially produced in tip cells that reside close to a high VEGF concentration. Endothelial cells at tips of new tubes compete for leader positions. They can suppress the Notch-restricted, tip-cell phenotype of their immediate neighbors when they synthesize DLL4 and present DLL4 to adjacent migrating cells [1394]. Many other primary messengers participate in blood vessel branching such as guidance molecules (netrins, semaphorins, and slits).

In developing lungs, branching is bounded by a volume of mesenchymal tissue. The bronchial tree indeed develops by branching of the airway epithelium into a surrounding mesenchyme. Reciprocalfeedbacks between the airway epithelium and surrounding mesenchyme control the normal sequence and three-dimensional pattern of branching events [1392].

The branched tree can be generated by a genetically tractable, hierarchical, and modular program that contains 3 local modes of branching used in 3 different orders, i.e., at different times: domain branching at the branching onset and planar and orthogonal bifurcations [1392]. These branching modes may arise from the combination of a few elements, i.e., a master branch generator and 3 genetically encoded slaves that govern the pattern of branching events [1392]: (1) a domain specifier; (2) a genetically programmed periodicity generator that determines when side branches arise; (3) a bifurcator that defines branching loci, i.e., their occurrence time; and (4) a rotator that directs the bifurcation angle w.r.t. the stem plane. These branching modules are characterized by their coupling and repetitive occurrence.

A primary conduit constructs branching domains that serve as scaffolds. Branching arises at different positions directed by the periodicity generator to give rise to 2 coplanar branches, one of which undergoes domain branching and the other bifurcates again in a planar manner. Planar bifurcation, i.e., splitting of a branch tip into 2 branches, generates most ternary and some quaternary branches. Orthogonal bifurcation involves planar bifurcation with rotation of the bifurcation plane to give rise to higher-order generations.

Several types of sequences arise according to branching modes [1392]. In sequence 1, a founder branch formed by domain branching uses immediately and permanently the orthogonal branching mode. In sequence 2, a founder branch formed by domain branching utilizes domain branching for some daughter tubes that then employ permanently the orthogonal branching mode. The founder forms other daughter ducts by planar bifurcation that themselves undergo planar bifurcation at their tips to form domain branches along their length. These domain branches switch permanently to orthogonal bifurcation. Other sequences can be observed.

However, branching pattern can vary [1392]. Branches can originate off an unusual parent branch. A branch can miss and daughter branches spring directly from the grandparent conduit. In addition, branching network size and architecture differ among species.

In mice, branches form in 4 domains: lateral, dorsal, medial, and ventral [1392]. The first secondary branch from the left primary bronchus lineage buds off the lateral sprout of the founder branch. Over the next 2 days, additional branches sprout distal to the first secondary branch and create a row of lateral secondary branches. Then, another row begins to form along the dorsal surface of the left primary bronchus. As this domain develops, a third row starts to sprout from the medial surface of the left primary bronchus. Afterward, a fourth domain eventually arises from the ventral surface. This ventral domain often consists of a single distal branch. Secondary branches off the distal portion of the right primary branch also arise by domain branching, beginning with a row of lateral branches. The branch spacing in each row is similar to the left pattern, but neither the row-initiation positions nor branch number in each row are the same.⁷

In domain branching, the main secondary daughter branches form in domains at different positions around the wall circumference of the parent branch, like rows of bristles on a bottle brush [1392]. However, each row is characterized by an independently patterned domain. A patterning controller of domain branching that directs the branching situation (either proximal or distal) is constituted of a periodicity generator that regulates the sequence of branching within each domain. A circumferential patterning controller specifies the positions of domains and the order with which domains are used.

Many ternary branches are constructed by domain branching. Nonetheless, some ternary and higher-order generations arise from a different mode of tip expansion and bifurcation. Planar bifurcation corresponds to branching with similar orientation w.r.t. the founder branch, such as that of certain ternary and quaternary branches [1392].

On the other hand, some ternary and most higher-order generations use a third branching mode, the orthogonal bifurcation. Branches bifurcate as in planar bifurcation, but between each round of branching with an approximately 90-degree rotation of the bifurcation plane. The daughter tubes are thus arranged in a rosette [1392].

Lung development relies on epithelial–mesenchymalfeedback loops that usegremlin-1,bone morphogenetic protein BMP4,fibroblast growth factor, as well assonic Hedgehog andWnt morphogens, andretinoic acid. Many transcription factors, such as zinc finger protein, multitype ZFPM2,⁸GATA4, andNKx2.1, are implicated in pulmonary branching [1394]. Components of the extracellular matrix, such as fibronectin and laminin, are also involved [1401].

Fibroblast growth factor secreted from mesenchymal cells near to epithelial tip cells, especially FGF10 that targets predominantlyFGFR2b receptor on emerging branch epithelial cells, promotes epithelial budding, outgrowth, and branching. Signaling launched by FGF10 provokes localsprouty-2 production at the branch tips [1392]. Sprouty-2 hinders themitogen-activated protein kinase modules and limits the proliferation and migration of tip cells, hence prohibiting further branch extension, but initiating new branching. Sprouty-2 can then be associated with the periodicity generator [1392]. An elevated activity of the periodicity generator reduces interbranch length and provokes a shift from bifurcation to trifurcation.

The signaling pathways mediated by hypoxia-inducible factor and vascular endothelial growth factor cooperate with the FGF–FGFR–sprouty-2 pathway to match the developing capillary network to the growing airway tree, thus optimizing gas-exchange capacity of lungs [1401]. Moreover, FGF upregulates expression of the Bmp2 and Bmp4 genes in tip cells. Agent FGF10 may also act as a chemoattractant [1394].

Bone morphogenetic protein BMP4 as well as sonic Hedgehog signaling by tip cells exert an autocrine regulation that restrict FGF-mediated branching. On the other hand, BMP4 synthesized by mesenchymal cells enhances branching (paracrine regulation) [1394]. Factor BMP4 favors proliferation and survival of lung epithelial tip cells.

Sonic Hedgehog regulates the progression rather than initiation of airway branching [1394]. It inhibits near-tip mesenchymal FGF10 synthesis, hence new branching in non-allocated loci. Therefore, sonic Hedgehog contributes to the structural reproducibility.

Wnt morphogen uses its canonical pathway to reinforce FGFR2b expression in epithelial cells [1394]. On the other hand, non-canonical Wnt5 signaling precludes FGF10 synthesis.

Cardiac stem cells divide either symmetrically or asymmetrically.¹¹ Myocardium contains stem cell niches [1409]. Cardiac stem cells are smaller then mature cardiomyocytes. Cardiac stem cells and supporting cells are connected by gap and adherens junctions. Integrins of primitive and committed cells within the niche attach these cells to extracellular matrix proteins (fibronectin and laminin) that transduce mechanical signals for differentiation regulation.

Stem cells may regenerate the myocardium, especially after myocardial infarction and in ischemic cardiomyopathy, because the heart is a weakly regenerative organ. Progenitor cells secrete paracrine factors and may contribute to vasculogenesis and tissue repair and remodeling [1410].

In the heart, resident stem cells can lead to endothelial and smooth muscle cells as well as cardiomyocytes [1411]. The heart may contain various populations of resident myocardial progenitors with different expression modes that may differentiate into cardiomyocytes and endothelial cells.¹²

Although cardiogenic progenitor cells maintain myocardial turnover, they do not lead to heart regeneration. Consequently, injured adult hearts form scars without cardiomyocyte proliferation. Appropriate differentiation of stem cell and induction of the division cycle of resident cardiomyocytes can be stimulated with suitable factors. Cytokine treatment (e.g., colony-stimulating factor CSF3) improves infarction repair, but can also increase mortality [1412]. Cell delivery can be done either by intracoronary infusion or direct intramyocardial injection during grafting or using endovascular procedures. Periostin activates plasmalemmal α_V-, α₁-, α₃-, and α₅-integrins, and induces re-entry of cardiomyocytes into the cell cycle [1413].

Circulating endothelial progenitor cells (EPC) promote neovascularization; hence, they are also calledcirculating angiogenic cells. They are reduced in number. They can become dysfunctional in chronic diseases. Circulating progenitors contribute only sligthly to cardiomyocyte repopulation. Endothelial progenitors can induce myogenesis in vitro, and angiogenesis for O₂ supply. A time window exists for optimal cell therapy. Infusion of EPCs promotes neovascularization after ischemia. Peptidasecathepsin-L expressed in EPCs (but not strongly in endothelial cells) degrades the matrix, thereby allowing invasion by EPCs [1414].

Skeletal myoblasts are quiescent stem cells with proliferative capacity in vitro and ischemic tolerance in vivo. Moreover, the risk of aberrant differentiation is limited [1415]. Unfortunately, implanted myoblasts do not necessarily differentiate into excitable cardiomyocytes. In particular, they are unable to couple electromechanically with cardiomyocytes. Skeletal myocytes actually do not express adhesion and junction proteins required for electromechanical coupling.

Transplantation of bone marrow cells possibly enriched with hematopoietic stem cells can generate coronary endothelium to a greater extent than myocardium [1412]. Bone marrow cells also enhance infarct healing. Works on bone marrow-derived mononuclear cells fail to demonstrate efficiency.

Adult bone marrow cells retain dedifferentiation capacity. They can enter into cardiomyocyte and vascular lineages. Bone marrow cells engraft, survive, and grow within the myocardium and form junctional complexes based on connexin-43 and N-cadherin with resident cardiomyocytes [1416]. Implantedhematopoietic stem cells differentiate into blood cells, but not significantly into cardiomyocytes.

Mesenchymal stem cells (MSC) of the stromal region (non-hematopoietic compartment) of the bone marrow produce growth factors and cytokines. They colonize sites of injury. Implanted MSCs decrease pathological wall remodeling. Bone marrow mesenchymal stem cells can differentiate into cardiomyocytes and vascular cells [1417], but they can have limited survival after transplantation in the myocardium [1418]. Furthermore, cell therapy may be ineffective or even hazardous in certain clinical settings and specific subgroups of patients [1419, 1420].

Bone marrow-derived mesenchymal stem cells that overexpress the Pkb1 gene (that encode PKB1 kinase) are better for cell therapy of acute myocardial infarction. Secretion of paracrine cytoprotective factors from stem cells exerts an effect on cardiomyocytes exposed to hypoxia [1421].

A mixed ester of hyaluronan with butyric and retinoic acid acts as a cardio- and vasculogenic agent in humanmesenchymal stem cells isolated particularly from fetal membranes of term placenta. Hyaluronan with butyric and retinoic acids primes stem cell differentiation into endothelial and cardiac cells [1422].

Embryonic stem cells differentiate into all cell types, especially cardiomyocytes organized in myofibers when stimulated by suitable cardiac promoters, but they can produce teratomas. Moreover, they can form ectopic pacemakers.

Human embryonic stem cells can differentiate to cardiomyocytes using activin-A and BMP4 factors. However, a mixed cell population with cardiomyocytes, endothelial cells, and fibroblasts must be cultured in a rotating orbital shaker to create human, functional, prevascularized, cardiac tissue patches [1423]. Preformed microvessels can anastomose with the host coronary circulation.

In summary, transdifferentiation has stimulated multiple clinical trials. Cardiac progenitors from the cardiac population and cardiomyogenic cells derived from adult bone marrow stromal cells are able to differentiate into cardiomyocytes when interacting with cardiomyocytes. Regenerative cardiology trials most often use bone marrow cells and mesenchymal stem cells, because cardiac stem cells do not generate significant repair. However, the results of stem cell injection in hearts after heart attack at most show slight, transient benefit. Cell treatment optimization hence remains challenging, taking into account disease type, patient history, associated therapy for the selection of the cell type, associated growth factors, delivery route, and administration timing.

Engineered heart tissue can form a thick myocardium after implantation on myocardial infarcts in immune-suppressed rats [1432]. When evaluated 28 days later, engineered heart tissue shows neither undelayed electrical coupling to the native myocardium, arrhythmia, nor dilation. Cardiomyocytes seeded onto mechanically conditioned, perfused, porous scaffolds can conduct action potentials and beat synchronously, but the scar hinders a proper integration of implanted cells.

Cell sheet technology applied to a monolayer of adipose tissue-derived, pluripotent,mesenchymal (non-hematopoietic) stem cells allows the repair of scarred myocardium after myocardial infarction in rat hearts [1433]. Engrafted mesenchymal stem cells prevent wall thinning in the scar area and improve the cardiac function.

Composite matrices can be used for heart valve engineering. Enzymatically decellularized porcine aortic valves that are impregnated with biodegradable polymer by a stepwise exchange provide a scaffold for heart valve tissue engineering with complete endothelialization [1434]. Ovine cell seeding of decellularized porcine heart valves (with almost complete preservation of the extracellular matrix) is improved under pulsatile flow conditions [1435].

Preliminary numerical experiments have been carried out to study the content and orientation of collagen fibers²⁴ of the loaded aortic valve [1437]. In an isotropic cusp, the fiber orientation is driven by the principal strain directions. The remodeling depends on the transvalvular pressure, fiber stiffness, initial fiber direction (close to the principal strain directions in this work), and loading conditions.

Sheet-based tissue engineering, using fibroblasts and endothelial cells, without smooth myocytes and exogenous biomaterials, can form cylindrical blood vessels, characterized by a composite multilayered wall with vasa vasorum [1438].

Arterial stenoses and lumen blockage by clots locally or emboli remotely can be treated by bypass grafting surgery. Autologous grafts (internal mammary artery, saphenous vein) are used in most patients. Synthetic grafts can also be utilized. To reduce the failure rate of synthetic (polytetrafluoroethylene) grafts due to thrombosis and scar within graft lumen, biocompatible, durable biosynthetic grafts have been made. Synthetic tubes are coated by an adhesion matrix (fibronectin) and endothelial cells on its interior surface. Endothelial cells are genetically changed to overexpressfibulin-5 in order to improve their adhesion on matrix and avoid flow-induced detachment [1439, 1440]. Because of their decayed capacity of proliferation, these modified endothelial cells are also genetically altered to overexpress vascular endothelial growth factor. A coating of adhesion matrix on the tube exterior surface allows seeding of smooth myocytes, which are also genetically altered to overexpress adhesion factors and growth factors.

Mesenchymal stem cells can also be used for the construction of vascular grafts. Strain influences the differentiation (particularly into smooth myocytes) and proliferation of mesenchymal stem cells, mechanotransduction depending on the orientation of cells with respect to the strain axis [1441].²⁵ The implicated signaling pathway can be affected by the activity of the cytoskeleton and focal adhesions, both subjected to applied loading.

A better understanding of mechanotransduction processes can improve the tissue engineering setup. Numerical simulations, once validated, are important because they: (1) provide the fields of imposed stresses and (2) can optimize the bioreactor design.

Lung design is characterized by an efficient structure–function relationship. The pulmonary architecture enables ventilation, perfusion, matching of these 2 functions, and gas exchange across an optimal gas transfer surface area. The anticoagulant and -immunogenic air–blood interface is immersed in a deformable pump. Because lung tissue has a limited regeneration capacity in vivo, lung transplantation is used in some circumstances, but is limited by a shortage of donor organs. In the case of bone marrow, successful transplantation relies on tissue devitalization followed by reintroduction of a regenerating cell population. After complete decellularization of isolated adult rat lungs, the acellular extracellular matrix that preserves the hierarchical branching structure of its vasculature and airways, i.e., that keeps scaffolds of vasculature, airways, and alveoli, is reseeded with lung epithelial and endothelial cells of neonatal lungs and cultured in a bioreactor that retains the developmental environment of fetal lung [1442, 1443]. The resulting tissue contains alveoli, microvessels, and small airways with the appropriate cell types and rheology. After sevarl days, the construct can be perfused with blood and ventilated with physiological pressures.

Leukocyte extravasation is influenced by: (1) pro-inflammatory cytokines, such asinterleukins IL1α, IL1β, and IL6, andTNFα, among others, and (2) chemoattractants for myeloid cells, such as ^Nformyl methionyl leucyl phenylalanine (fMLP),complement component C5a,leukotriene LTb4, andplatelet-activating factor, produced in the inflammatory site [1445].

The secretion of a selective set of chemokines and upregulated expression of endothelial adhesion molecules caused by specific cytokines direct the subpopulations of leukocytes that are recruited during different phases of inflammation. In response to pro-inflammatory stimuli, such as TNFα, IL1β,Ifnγ, andthrombin,endothelial cells synthesize differentchemokines, such as CCL2, CCL5, CXCL1, CXCL8, CXCL10, or CXCL12 [1445].

Endothelial cells produce chemokines attached to glycosaminoglycans and Duffy receptor for chemokines, especially in apical endothelial microvilli [1445]. These endothelium-immobilized chemokines promote the transition from selectin-mediated tethering and rolling of leukocytes to integrin-mediated firm attachment and spreading, as they activate their cognate Gi/o-coupledchemokine receptors to express leukocyteintegrins. In addition,neutrophils secrete chemoattractants to recruit other leukocyte types in later stages of inflammation.

During inflammation, myeloid cell subpopulations, in which several chemokine receptors are upregulated, migrate from the bone marrow to inflammation loci and uptake antigens. The innate immune response that involvesmonocytes,macrophages, anddendritic cells, occurs in the inflamed tissue. Migration of immature dendritic cells to inflammation zones depends on the expression of chemokine receptors such as CCR2, CCR5, and CCR6 [1445]. After antigen uptake, the density of these chemokine receptors on mature dendritic cells decays, whereas CCR7 expression rises.

Conventional dendritic cells can then move via lymphatics to draining lymph nodes. In lymph nodes, mature dendritic cells present antigens to specific naive T cells. Naive T lymphocytes differentiate into effector(T_H1, T_H2, T_H9, T_H17, T_H22, and follicular T cells) andregulatory T lymphocytes (Table 11.5). Effector CD4 + T cells, T_Reg ,cytotoxic CD8 + T cells, andNK cells upregulate chemokine receptors to also migrate to inflammation sites and mount a specialized immune response. In addition, Ly6c^{high 29} Ly6g + ³⁰ monocytes migrate to participate in mucosal immunity [1445].

Numerousadhesion molecules are implicated in transmigration, such as: (1) selectins (E-, L-, and P-selectins);³¹ (2) cadherins such as vascular endothelial (VE)-cadherin, or cadherin-5; (3) immunoglobulin-like adhesion molecules, such as platelet–endothelial cell adhesion molecule PECAM1, intercellular adhesion molecules (e.g., ICAM1 and ICAM2), and vascular cell adhesion molecule VCAM1; (4) integrins, such as α₄β₁-, α₄β₇-, α_Lβ₂-, and α_Mβ₂-integrin; as well as (5) integrin-associated protein, or leukocyte surface antigen CD47 (Table 11.6; Vols. 1 – Chap. 7. Plasma Membrane and 2 – Chap. 6. Cell Motility). Leukocyte integrins connect to their endothelial ligands that are organized in tetraspanin-enriched microdomains [1445]. These cellular structures mediate the contact between leukocytes and endothelial cells during extravasation.

Endothelial adhesion receptors of activated endothelial cells localize to adhesion platforms that are enriched in tetraspanins in apical microvilli. Tetraspanins interact simultaneously with other tetraspanins and other types of transmembrane receptors. These adhesion platforms contain selectins and members of the Ig superfamily (VCAM1, ICAM1, ICAM2, PECAM1, and JAMs) [1445]. These functional units that coalesce around adherent leukocytes are connected to the actin cytoskeleton and contain α-actinin, ezrin, moesin, paxillin, talin, vinculin, and vasoactive stimulatory phosphoprotein. They are regulated by the Rho–RoCK pathway and PIP₂ second messengers. These docking structures, or transmigratory cups, stabilize firm adhesion of leukocyte to endothelium, hence preventing leukocyte detachment.

During leukocyte displacement over the endothelium, L-selectin, epican (CD44), ICAM1, and ICAM3, among others redistribute to the leukocyte rear pole. On the oher hand, integrins, such as α_Lβ₂– and α_Mβ₂-integrins, and chemokine receptors localize to the leading edge and endothelial contact area.³²

Leukocytes probe for sites suitable for diapedesis by constructing protrusions. When they find an appropriate site for migration, filopodia evolve into an invasive pseudopod to cross the endothelium. During paracellular transmigration, ICAM1 and JAMa interact with α_Lβ₂-integrin and form a ring-shaped cluster around the leukocyte [1445]. Transcellular migration requires the supply of additional membrane by the vacuolovesicular organelles to build a transcellular pore that lenghtens toward the basal membrane. Both ICAM1 and caveolin-1 translocate toward the transcellular pore. Membrane fusion is regulated by Ca^2 + and SNARE-containing complexes [1445]. Intermediate filament constituent vimentin contributes to the transcellular pore.

The selectin family of transmembrane adhesion receptors mediates leukocyte tethering and rolling. Selectin-P on endothelial cells is the primary adhesion molecule for cell capture and rolling onto vascular endothelium. The main leukocyte ligand for P-selectin is selectin-P ligand (SelPLg; a.k.a. P-selectin glycoprotein ligand-1 [PSGL1] and CD162).³³

Selectin-L also contributes significantly to leukocyte capture. Selectin-L and -E also take part in rolling. Selectin-L is much less efficient than P-selectin in cell rolling. Nonetheless, it is necessary to capture leukocytes and initiate their rolling. Selectin-E may be responsible for slow rolling interactions and possibly the initiation of firm adhesion.

Selectin-E and -P interact with L-selectin and PSGL1 expressed by leukocytes (Table 11.7). In addition, E-selectin targets other specific ligands, such as E-selectin ligand ESL1 (a.k.a. Golgi body sialoglycoprotein GlG1 and cysteine-rich fibroblast growth factor receptor CFR1), epican (a.k.a. extracellular matrix receptor ECMR3, GP90 lymphocyte homing–adhesion receptor, heparan sulfate proteoglycan receptor, and CD44).

β₂ Integrins participate in leukocyte deceleration and arrest. Neutrophils express small amounts of other integrins such as α₄β₁-integrin that binds to endothelial VCAM1 molecule. Integrin-α_Lβ₂ is the most important integrin in firm leukocyte adhesion, whereas α_Mβ₂-integrin seems to be important in neutrophil activation and phagocytosis. Both α_Lβ₂– and α_Mβ₂-integrins can bind to ICAM1 and ICAM2 molecules.

Neutrophils and some lymphocyte subpopulations use preferentially α_Lβ₂-integrin for firm adhesion. On the other hand, monocytes and lymphoblasts target predominantly α₄β₁-integrin [1445].

Several integrin ligands, such as vascular cell (VCAM1) and intercellular (ICAM1–ICAM2) adhesion molecules, are involved in firm adhesion, crawling, and paracellular and transcellular migration. Other types of Ig-like adhesion molecules, such as platelet–endothelial cell (PECAM1), junctional (JAM), and endothelial cell-selective adhesion molecule (ESAM), T-cell surface glycoprotein-E2 (or CD99), among others, act during paracellular transmigration [1445] (Table 11.8).