Myosin Heavy Chain (SMMHC) SM-B Isoform Is Differentially Expressed in Developing and Adult Smooth Muscle Tissues of the Rat

and atria l naturetic peptide; NEP blockade has been shown to reduce the development of hypoxia-induc ed pulmonary hypert ension . We e mbarked on a study to determine expression of a panel of peptidases (NEP, angiotensin-converting enzyme [ACE ], and aminopeptidase N [APN]) in primary pulm ona ry hyperten sion (PPH) compared to the expression in the lungs of patients with intestitial lung disease (ILD) and norm al subjects. Lun g tissue was stained with anti-NEP, AC E, and APN antibodies; staining intensity was grade d in the e ndothe lium (E N), media, and adventitia of small, mediu m, and large vessels and plexiform lesions, as well as in the alveolar epithelium. Adjacent sections were stain ed with antibody against factor VIII -related antigen to identi fy EN and to standardize the grading. Of note, APN immunostainin g was absent in ILD (n = 15), and rare focal stainin g « 1%) was seen in normal subjects (n = 8). However in PPH , APN was positive for 6 of 6 pat ients in the media and adventitia of small, medium, and large vessels, but for only 3 of 6 patients in the EN layer of small, medium, and large vessels; APN staining was present in the adventitia and media of the plexiform lesions for only 1 of 6 patients and was absent in the EN of the plexiform lesions for all 6 patients. NE P and ACE were present in all cell types in norm al subjec ts and in patients with ILD, but the staining distribution differ ed from PPI-I. NE P was see n in all cell types and vessels in PPH , with the exception that NEP staining in EN cells of plexiform lesions was abse nt in 8 of 9 patie nts (score = O.08), which was less than the NE P scores for media (1.4) and adventitia (3.94) of the plexiform lesions (p< O.OOOl) . ACE staining in plexiform EN was significantly less than in plexiform media (1.0) or adventitia (1.1) (p
low or absent in the EN of PPH plexiform lesions , APN is globally overexpressed in PPH as compared to normal subjects and patients with ILD. Peptidase inactivation may be part of the hyperten sive EN ph enotype. In this regard , pul mona ry plexiform lesion EN may resemble lung cancer cells, in which NE P is also inactivated .

Myosin Heavy Chain (SMMHC) SM-B Isoform Is Differentially Expressed in Developing and Adult Smooth Muscle Tissues of the Rat* Robert B. Low, PhD; and ShenJI L. Wh ite, PhD

(CHEST 1998; 11 4:31 S-33S)

T he smooth muscle myosin s ar e hexam eri c proteins

composed of two hea vy cha ins and two pairs of light chains. Four smooth muscl e myosin heavy chain (SMMHC) isoforms are known to exist (SM-I A, SM-IB , SM-2A, and SM-2B ),1.2 and th eir exp ress ion is restricte d to smo oth mu scle. Th e patt ern of express io n of two isoform s, SM-IA and SM-2A, ha s been descr ib ed for a nu mb er of vasc ular an d visceral smoo th mu scle tissues. Smoo th muscl e isoform conte nt varies during development and in th e adult, including th e hm g.:3 Th ough expec ted from studies of cardiac and skele tal muscle myosin heavy chain Isoforms.s" result s from many labo ratories to date have not found a clear relationship between the proportions of SM-I A and SM-2A and tissue contractile properti es." This may be du e to the fact that the differ en ce between these two isoforms resides in the "tail" region of the heavy chain molecule, far removed from the head region that possess adenosin e triphosphatas e and actin-binding activity. Th e SM-B isoforms differ in th e head region , in a fashion that is known to affect in vit ro enzyme and actin-binding activities.t -? In particul ar , th e seven amino acid insert in th e SM-B isoform s is both necessary and sufficie nt to incr eas e in vitro ade nosine triphosphatase activity and velocity of movem ent in an in vitro motility assay.' Earlier studi es ba sed on messen ger RN A (mRNA) ana lyses suggested th at th e SM- B isoform may be pr esent se lec tively in fast ph asic vs slow toni c smooth m uscle .I-"

We have now used mon ospecific polyclon al antibodies to localize th e SM-B isofo rm in developing rat smooth mu scle tissues, including the lun g. For making SM -B-specific antibo dy, th e SM- B di fferen ce peptide, G ln-G ly-Pro -Ser-Ph e- Ala-Tyr -Gly-Gln- Leu -Gin -Cys, was co upled to keyhole limp et hemocyanin at th e cysteine residu e and inject ed int o rabbits (HT I BioSer vices, In c; Boston ). Specificity was dem on strated by FIG UR E 1. Alveolar epithe lium and endothelial cell staining in PPH patients.

"Fr om th e Depart ment of Molecular Physiology and Bioph ysics, Univer sity of Ver mont , Burlington. CHEST /114 /1 / JULY, 1998 SUPPLEM ENT

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FIGURE 1. lmnumohi xtrx-liemi cal sta ining of IlIlIg hlood ves se ls. Three diff erent sizes of ves se l ar e stained wit h untihody that reeogllizes SM -B (left) or all smooth muscle myosin heavy chalns (right). (O Jiginal magllineatioll X400; har = 10 u.m ).

immunoblot against a tis su e pan el (b rain, cardiac muscle , skel etal muscle, lung, stomach , b ladder, intestin e , ut erus ), by tissue distribution (iun u unohistochc mist ry), and by reactivity to baculoviru s exp re sse d myosin head fra gm ent with and without th e sev en amino acid SM-B ins ert. (Antibody that recognizes all SMMIIC was provid ed by Dr. Robert Adelstein , National Institutcs of H ealth , Bethesd a, Md .) Ribonucl ease protection assays were carried out as d es cribed ." For immunohistoch emistry, tissu es were fixed in 100 % e thano l, se ct ion ed, and prepared for immunostain ing in co nve ntio na l fashion . Tis su es were in cubated with primury antibod y, followed by biotinylated goat an tira bhi t se condar y antibody, and th en st re ptavid in-pe roxid asc. Detection wa s with diaminobcnzidine . SM -B mHNA predominates over SM-A mHNA (lacks ins ert ) in neonatal aorta but its exp re ssio n diminishes 325

co ns ide rab ly with d ev e lopm ent , SM -A mHNA being th e predominant message in th e adult. SM-B mRNA dominates in th e bladder in d ev elopm ent and in th e adult, while in stomach and int estin e , SM -B is prominent in dev elopm ent but SM-A mHNA becom es e q ual or slightly g re ate r in th e ad ult. In lun g, both SM -B and SM-A mRNAs a re pres ent durin g d evelopm ent starting around birth , while SM-A mH [A predomin at es in th e adult. By Western blot anal ysis, SM- B protein exp re ssion ge ne rally follows SM-B mHNA in most organs of th e body. At th e same tim e, th e proportions of mHNA to protein su ggest differences in d et ection se nsitivity and/or significant po sttranscriptional regulation . During lung d ev elopm ent, SM-B protein first is d ete ct ed immunohistochemically in th e trachea and bronchi at th e saccular stage of d e velopment around th e

Thomas L. Petty 40th Annual Aspen Lung Conference: Bio logy & Pathobiology of the Lung Circu lation

tim e of bi rth, wh en other smooth muscle myosin hea vy ch ain isoforms also are known to b e present." Thereafte r, reactivity sp reads di st ally through th e airways, reaching th e level of alveolar se ptae in th e adu lt. No reacti vity is seen in th e major blood ves se ls, but resistan ce ves sels b ecome highly rea ctiv e b eginning approximately 3 week s aft e r b irth . Cha rac te ristic ally, th ere is marked het erogeneity of SM -B staining of sm all blood vess els relative to th e p attern seen with myosin h ea vy cha in antibo dies that reco gnize all sm ooth muscle myosins (F ig 1). Th e distribution of th e SM-B isoforms in th e smooth mu scle tissues of the bod y suggests that th ey are found predominantly in fast ph asic smooth mu scle tissu es. We hypothesize th at in the lun g, SM-B plays an important role in regulation of contractile fun ction of th e airways and resistan ce vasculature, for example, in controlling ventilation and perfusion. REFERENCES 1 Rovner AS, Freyzon Y, Trybus KM. An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms. J Muscle Res Cell Motil 1997; 18:103-10 2 White S, Martin A, Periasamy M. Identification of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the SI head region. Am J Physiol 1993; 264: C1252-55 3 Woodcock-Mitchell J, White S, Stirewalt \V, et al. Myosin isoform expression in developing and remodeling rat lung. Am J Respir Cell Mol BioI 1993; 8:617-25 4 Swynghedauw B. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev 1989; 66:710-71 5 Somlyo A. Myosin isoforms in smooth muscle: how may they affect function and structure. J Muscle Res Cell Motil1993; 14:557-63 6 Seidel C, Rickman D, Steukrath H , et al. Control and function of alterations in contractile protein isoform expression in vascular smooth muscle. In: Moreland RS, ed. Regulation of smooth muscle contraction, advances in expe ri me ntal medicine and biology New York: Plenum Press, 1991; 304:315-25 7 Kelley CA, Takahashi M, Yu JH, et al. An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. J BioI Chern 1993; 268:12848-54

Hypoxia Upregulates Inducible (Type II) Nitric Oxide Synthase in an HIF-1 Dependent Manner in Rat Pulmonary Microvascular But Not Aortic Smooth Muscle Cells* Lisa A. Palmer, PhD; Roger A. Johns, MD

(CHEST 1998; 114:33S-34S)

Chronic hypoxia in th e pulmonary vasculature results in vasc ular remodeling chara cte rize d by prolife ration and migr ation of smooth muscle ce lls. Several factors induced by hypoxia ha ve been implicated as modulators or mediators in th e vascular remodeling process in hypoxia-induced pulmonary hyp ertension . O f th es e , nitric oxide is a sh ort-lived inter- and intracellular second me ssenger ge ne rate d by a family of enzy mes known as nitric oxide synthases (N O S). Th e mech anism by wh ich Typ e II (ind uc ib le) NOS ge ne exp re ssion is regulated by hypoxia is an ac tive are a of in vestigation . Hypoxic regul ation of th e Type II NOS ge ne require s a cis-acting transcription fact or known as hypoxic inducibl e factor-1 (H I F -1).1 This transcription facto r is Wid ely exp re sse d in a number of ce ll typ es,> ye t th e response of a particular ge ne to hypoxia is dependent on cell type." Previous stud ie s from our laboratory have demonstrated an upregulation of Type II NOS in th e vascular smooth muscle of rat pulmonary arte ry and microvess els aft er chronic hypoxia.r> Th e obj ective of th e curre nt study was to co mp are hypoxia regulation of Type II NO S in rat pulmonary microvascular smooth muscle (mVSM) ce lls to aortic vasc ular smooth muscle (aVSM) cells and d et ermine wh ether HIF-1 is involved in this regulation . The rat mVSM cell s were obtained and ch aracterized by th e method of Johnson et al," whil e th e aVSM ce lls were isolate d and ch aracterized as p reviously des cr ib ed." Transi ent tran sfection studi es wer e performed in mVSM and aVSM cells und er hypoxic (pOz of 14.9::':::1.2 mm Hg for 48 h ) and normoxic conditions using the 5' -flanking region of the Type II NOS gene linked to the reporter gene, chloramph enicol acetyltransfer ase (p1iNOSCAT). In mVSM cells, p1iNOSCAT activity was increased 2.2::'::: 0.7-fold in hypoxiaexposed cells, as compared with norm oxia-exposed cells. Del etion (p220) or mutation (p209) of the I-IIF-1 binding site completely abolished tins hypoxia-indu ced increase in Type II NOS promot er activity. In contrast, hypoxia failed to increase p1iNOS CAT activity in aVSM cells (Table 1). El ectromobility shift analysis was performed on nuclear extracts made from aVSM and mVSM ce lls grown for 14 h and 4 h , resp ectively, in normoxia or hypoxia and a 30-base pair oligonucleot ide containing th e HIF-1 binding site present in the 5 ' -flanking region of th e Typ e II NOS ge ne . HIF-1 binding activity was induced by hypo xia in both aVSM and mVSM ce lls. Binding activity of this complex was spe cific for th e HIF-1 sequen ce as incr easing molar excess of th e HIF-1 oligonucleotide effec tively compe te d for binding activity, whe reas compe tition with an oligonucleotide mutated at residues known to be necessary for binding HIF-1 protein had no effect. To det ermine if hyp oxia induces th e exp re ssion of th e endogenou s Type II NOS ge ne, aVSM and mVSM ce lls were grown under no rm oxic or hypo xic cond ition s in th e absence or presen ce of 300 nglmL lipopolysacch a'From the Department of Anesthesiology, University of Virginia, Charlottesville.

Reprint requests: Lisa A. Palmer, PhD, Dept of A nesthestologi], University of Virginia, PO Box 100]0, Charlottesville, VA 229060010 CHEST / 114 / 1 / JULY , 1998 SUPPLEMENT

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